Cyclic tetrapeptides and therapeutic applications thereof

ABSTRACT

There are provided compounds of formula I wherein k, m, n, p, R, R′, R″, R′″, R3 and R4 are as defined in the application. Other embodiments are also disclosed.

BACKGROUND

Immunosuppressive drugs are commonly used in transplantation and intreatment of autoimmune diseases. Production of these drugs isexpensive, and the most frequently used of these drugs, namelycyclosporine A, tacrolimus and rapamycin, exhibit undesirableside-effects. The search for new immunosuppressive drugs devoid ofside-effects, particularly in the class of natural peptideimmunoregulators and their analogues, represents a serious challenge formedicinal chemistry.

Cyclolinopeptide A (CLA), a very hydrophobic cyclic nonapeptide, wasfirst isolated from linen seeds in 1959. CLA is stronglyimmunosuppressive, with a potency comparable to that of cyclosporine A(CsA). The mechanism of action of CLA was shown to be similar to that ofCsA, i.e. CLA formed a complex with cyclophilin A causing inactivationof calcineurin, albeit at much lower affinity (Gaymes et al., Febs Lett,1997, 418, 224-227). CLA inhibited both humoral and cellular immuneresponse and graft-versus-host reaction; prolonged survival ofallogeneic skin grafts; tempered post-adjuvant polyarthritis in rats andhemolytic anemia of New Zealand Black mice; and, similarly to CsA,inhibited IL1 and IL-2 production. Unfortunately, the highhydrophobicity of CLA presents an obstacle for the potential applicationof the compound in therapy.

Linear CLA analogues containing alanine residue in successive positionsof the peptide chain were found to be immunosuppressive (Wieczorek etal., Arch Immunol Ther Exp, 1992, 40, 213-216). It was also found thatthe activity of linear CLA analogues gradually decreased with shorteningof the peptide chain from the N-terminus, at the same time showing anincrease of activity for C-terminal tetra- and tripeptides (Siemion etal., Arch Immunol Ther Exp, 1994, 42, 459-465). The introduction of asingle, hydrophilic threonine residue into the CLA molecule did notresult in improved solubility in water. However, an improvement insolubility was achieved by the introduction of a sulphonic group in thepara-position of the phenyl ring of one or two phenyloalanine residues,without loss of biological activity (Siemion et al., Arch Immunol TherExp, 1992, 40, 257-261; Cebrat et al., J Peptide Res., 1997, 49,415-420). In addition, it has been observed that the inclusion oftetrapeptidic (Pro-Pro-Phe-Phe) or tripeptidic (Pro-Phe-Phe) fragmentsin longer linear peptides chains seem to have significance forimmunosuppressive activity (Wieczorek et al., Arch Immunol Ther Exp,1993, 41, 291-296; Cebrat et al., Pol. J Chem, 1997, 71, 1401).

A series of analogues in which the cis-peptide bond between prolineresidues was replaced with 1,5-disubstituted tetrazole ring (a goodmimetic of amide bonds in cis configuration) showed immunosuppressiveactivity comparable to CsA. (Karczmarek et al., Biopolymers, 2002, 63,343-357).

Synthetic CLA analogues in which leucine residues in position 5 and/or 8were replaced with their hydroxymethyl analogue displayed a four-foldincrease in solubility in water in comparison to CLA, but also showed a25% diminution in biological activity compared to native CLA (Zubrzak etal., Biopolymers (Peptide Science), 2005, 80, 347-356).

A series of nine CLA analogues was obtained by replacement of CLAproline residues with β²-isoproline and β³-homoproline. In comparison toCsA, these CLA analogues displayed strong inhibitory properties in thecellular immune response. The majority of these analogs were practicallydevoid of cell toxicity (Katarzyńska et al., J Pept Sci, 2009, 14,1283-1294).

BRIEF DESCRIPTION OF THE INVENTION

There are provided in accordance with an embodiment of the presentinvention compounds having the formula I:

wherein

k, m, n and p are each independently 0, 1 or 2;

R and R′ are each independently selected from H and C₁₋₃ alkyl, or, whentaken together, R and R′ are —CR¹R^(1′)—X—CH₂—, wherein CR¹R^(1′) isattached to the backbone nitrogen, R¹ and R^(1′) are each independentlyselected from H and C₁₋₃ alkyl, and X is selected from —CH₂—, —CH₂CH₂—,—CH(OH)—, —O—, —S— and —NH—;

R″ and R′″ are each independently selected from H and C₁₋₃ alkyl, or,when taken together, R″ and R′″ are —CR²R^(2′)—X′—CH₂—, whereinCR²R^(2′) is attached to the backbone nitrogen, R² and R^(2′) are eachindependently selected from H and C₁₋₃ alkyl, and X′ is selected from—CH₂—, —CH₂CH₂—, —CH(OH)—, —O—, —S— and —NH—; and

R³ and R⁴ are each independently selected from aryl, substituted aryl,heteroaryl and substituted heteroaryl;

or a pharmaceutically acceptable salt thereof.

In some embodiments, at least one of R³ and R⁴ is phenyl. In someembodiments, at least one of R³ and R⁴ is 4-hydroxyphenyl. In someembodiments, at least one of R³ and R⁴ is 4-t-butoxyphenyl. In someembodiments, at least one of R³ and R⁴ is 2-indolyl. In someembodiments, R³ and R⁴ are both phenyl. In some embodiments, one of R³and R⁴ is phenyl and the other of R³ and R⁴ is 4-hydroxyphenyl. In someembodiments, one of R³ and R⁴ is phenyl and the other of R³ and R⁴ is4-t-butoxyphenyl. In some embodiments, one of R³ and R⁴ is phenyl andthe other of R³ and R⁴ is 2-indolyl. In some embodiments, the carbon towhich —CH₂—R³ is attached has absolute (R)-stereochemistry. In someembodiments, the carbon to which —CH₂—R³ is attached has absolute(S)-stereochemistry. In some embodiments, the carbon to which —CH₂—R⁴ isattached has absolute (R)-stereochemistry. In some embodiments, thecarbon to which —CH₂—R⁴ is attached has absolute (S)-stereochemistry. Insome embodiments, one of k, m, n and p is 1, and the remainder of k, m,n and p are 0. In some embodiments, two of k, m, n and p are 1, and theremainder of k, m, n and p are 0. In some embodiments, at least one of kand m is not 0. In some embodiments, at least one of n and p is not 0.In some embodiments, at least one of k and m is not 0 and at least oneof n and p is not 0. In some embodiments, both k and n are 0. In someembodiments, both k and n are 0, one of m and p is 0, and the other of mand p is 1. In some embodiments, both k and n are 0 and both m and pare 1. In some embodiments, all four amino acids are L-amino acids. Insome embodiments, three of the amino acids are L-amino acids and one ofthe amino acids is a D-amino acid. In some embodiments, two of the aminoacids are L-amino acids and two of the amino acids are D-amino acids. Insome embodiments, one of the amino acids is an L-amino acid and three ofthe amino acids are D-amino acids. In some embodiments, all four aminoacids are D-amino acids.

In some embodiments, R and R′ are taken together to form —(CH₂)₃—, i.e.R and R′ are taken together to form —CR¹R^(1′)—X—CH₂— wherein R¹ andR^(1′) are both H and X is CH₂. In some embodiments, R″ and R′″ aretaken together to form —(CH₂)₃—, i.e. R″ and R′″ are taken together toform —CR²R^(2′)—X′—CH₂— wherein R² and R^(2′) are both H and X′ is CH₂.In some embodiments, R and R′ are taken together to form —(CH₂)₄—, i.e.R and R′ are taken together to form —CR¹R¹—X—CH₂— wherein R¹ and R^(1′)are both H and X is (CH₂)₂. In some embodiments, R″ and R′″ are takentogether to form —(CH₂)₄—, i.e. R″ and R′″ are taken together to form—CR²R^(2′)—X—CH₂— wherein R² and R^(2′) are both H and X is (CH₂)₂. Insome embodiments, R and R′ are taken together to form —CH₂—CH(OH)—CH₂—,i.e. R and R′ are taken together to form —CR¹R^(1′)—X—CH₂— wherein R¹and R^(1′) are both H and X is CH(OH). In some embodiments, R″ and R′″are taken together to form —CH₂—CH(OH)—CH₂—, i.e. R″ and R′″ are takentogether to form —CR²R^(2′)—X′—CH₂— wherein R² and R^(2′) are both H andX′ is CH(OH). In some embodiments in which R and R′ are taken together,the carbon at which R′ is attached has absolute (S)-stereochemistry. Insome embodiments in which R and R′ are taken together, the carbon atwhich R′ is attached has absolute (R)-stereochemistry. In someembodiments in which R″ and R′″ are taken together, the carbon at whichR′″ is attached has absolute (S)-stereochemistry. In some embodiments inwhich R″ and R′″ are taken together, the carbon at which R′″ is attachedhas absolute (R)-stereochemistry.

In some embodiments, the compound is selected from the group consistingof:

In some embodiments, the compound is a compound of formula I-1. In someembodiments, the compound is a compound of formula I-2. In someembodiments, the compound is a compound of formula I-3. In someembodiments, the compound is a compound of formula I-4. In someembodiments, the compound is a compound of formula I-5. In someembodiments, the compound is a compound of formula I-6. In someembodiments, the compound is a compound of formula I-7. In someembodiments, the compound is a compound of formula I-8.

In some embodiments, the compound is selected from the group consistingof:

In some embodiments, the compound is a compound of formula I-A. In someembodiments, the compound is a compound of formula I-B. In someembodiments, the compound is a compound of formula I-C. In someembodiments, the compound is a compound of formula I-D. In someembodiments, the compound is a compound of formula I-E. In someembodiments, the compound is a compound of formula I-F. In someembodiments, the compound is a compound of formula I-G. In someembodiments, the compound is a compound of formula I-H. In someembodiments, the compound is a compound of formula I-I. In someembodiments, the compound is a compound of formula I-J. In someembodiments, the compound is a compound of formula I-K. In someembodiments, the compound is a compound of formula I-L. In someembodiments, the compound is a compound of formula I-M. In someembodiments, the compound is a compound of formula I-N. In someembodiments, the compound is a compound of formula I-O. In someembodiments, the compound is a compound of formula I-P.

In some embodiments, one or more amino groups in the compound of FormulaI are in protected form.

There is also provided, in accordance with an embodiment of theinvention, a pharma-ceutical composition comprising a compound offormula I and a pharmaceutically acceptable carrier, excipient ordiluent therefor. In some embodiments, the compound is a compound offormula I-1. In some embodiments, the compound is a compound of formulaI-2. In some embodiments, the compound is a compound of formula I-3. Insome embodiments, the compound is a compound of formula I-4. In someembodiments, the compound is a compound of formula I-5. In someembodiments, the compound is a compound of formula I-6. In someembodiments, the compound is a compound of formula I-7. In someembodiments, the compound is a compound of formula I-8. In someembodiments, the compound is a compound of formula I-A. In someembodiments, the compound is a compound of formula I-B. In someembodi-ments, the compound is a compound of formula I-C. In someembodiments, the compound is a compound of formula I-D. In someembodiments, the compound is a compound of formula I-E. In someembodiments, the compound is a compound of formula I-F. In someembodi-ments, the compound is a compound of formula I-G. In someembodiments, the compound is a compound of formula I-H. In someembodiments, the compound is a compound of formula I-I. In someembodiments, the compound is a compound of formula I-J. In someembodiments, the compound is a compound of formula I-K. In someembodiments, the compound is a compound of formula I-L. In someembodiments, the compound is a compound of formula I-M. In someembodiments, the compound is a compound of formula I-N. In someembodiments, the compound is a compound of formula I-O. In someembodiments, the compound is a compound of formula I-P.

There is also provided, in accordance with an embodiment of theinvention, a method of suppressing immune response in a patient,comprising administering to a patient in need thereof an efficaciousamount of a compound of formula I. In some embodiments, the immuneresponse which is suppressed is inflammation. In some embodiments theimmune response which is suppressed is transplant rejection. In someembodiments, the compound is a compound of formula I-1. In someembodiments, the compound is a compound of formula I-2. In someembodiments, the compound is a compound of formula I-3. In someembodiments, the compound is a compound of formula I-4. In someembodiments, the compound is a compound of formula I-5. In someembodiments, the compound is a compound of formula I-6. In someembodiments, the compound is a compound of formula I-7. In someembodiments, the compound is a compound of formula I-8. In someembodiments, the compound is a compound of formula I-A. In someembodiments, the compound is a compound of formula I-B. In someembodiments, the compound is a compound of formula I-C. In someembodiments, the compound is a compound of formula I-D. In someembodiments, the compound is a compound of formula I-E. In someembodiments, the compound is a compound of formula I-F. In someembodiments, the compound is a compound of formula I-G. In someembodiments, the compound is a compound of formula I-H. In someembodiments, the compound is a compound of formula I-I. In someembodiments, the compound is a compound of formula I-J. In someembodiments, the compound is a compound of formula I-K. In someembodiments, the compound is a compound of formula I-L. In someembodiments, the compound is a compound of formula I-M. In someembodiments, the compound is a compound of formula I-N. In someembodiments, the compound is a compound of formula I-O. In someembodiments, the compound is a compound of formula I-P.

There is also provided, in accordance with an embodiment of theinvention, a method of treating or preventing an immune-mediated diseaseor condition in a patient, comprising administering to a patient in needthereof an efficacious amount of a compound of formula I. There is alsoprovided, in accordance with an embodiment of the invention, a methodfor lowering the toxicity profile of a second drug, comprisingadministering a compound of formula I in conjunction with the seconddrug. In some embodiments, the immune-mediated disease or condition isselected from the group consisting of auto-immune diseases,inflam-mation processes, transplant rejection, and allergic reactions.In some embodiments, the immune-mediated disease or condition isselected from Psoriasis, lichen planus and other papulosquamousdisorders. In some embodiments, the immune-mediated disease or conditionis selected from eczema and dermatitis. In some embodiments, the eczemeaor dermatitis is selected from eczema, atopic eczema, seborrheicdermatitis, and pompholyx. In some embodiments, the immune-mediateddisease or condition is a skin reaction to sunlight. In someembodiments, the immune-mediated disease or condition selected fromnon-specific skin irritation and insect bite. In some embodiments, theimmune-mediated disease or condition is a urticaria. In someembodiments, the immune-mediated disease or condition is selected fromthe group consisting of a primary skin tumor (e.g. melanoma); rheumatoidarthritis (both autoimmune and elicited by infection); Crohn's disease;inflammatory bowel disease; irritable bowel syndrome; aneurodegenerative disease (e.g. multiple sclerosis); Parkinson'sdisease; Graft-versus-Host reaction; severe psoriasis; and atopicdermatitis. In some embodiments, the compound of formula I isadministered in conjunction with a chemotherapeutic drug in order toreduce the toxic effects of the chemotherapeutic drug. In someembodiments, the compound is a compound of formula I-1. In someembodiments, the compound is a compound of formula I-2. In someembodiments, the compound is a compound of formula I-3. In someembodi-ments, the compound is a compound of formula I-4. In someembodiments, the compound is a compound of formula I-5. In someembodiments, the compound is a compound of formula I-6. In someembodiments, the compound is a compound of formula I-7. In someembodiments, the compound is a compound of formula I-8. In someembodiments, the compound is a compound of formula I-A. In someembodiments, the compound is a compound of formula I-B. In someembodiments, the compound is a compound of formula I-C. In someembodiments, the compound is a compound of formula I-D. In someembodiments, the compound is a compound of formula I-E. In someembodiments, the compound is a compound of formula I-F. In someembodiments, the compound is a compound of formula I-G. In someembodiments, the compound is a compound of formula I-H. In someembodiments, the compound is a compound of formula I-I. In someembodiments, the compound is a compound of formula I-J. In someem-bodiments, the compound is a compound of formula I-K. In someembodiments, the compound is a compound of formula I-L. In someembodiments, the compound is a compound of formula I-M. In someembodiments, the compound is a compound of formula I-N. In someembodiments, the compound is a compound of formula I-O. In someembodiments, the compound is a compound of formula I-P.

There is also provided, in accordance with an embodiment of theinvention, a kit, comprising a compound of formula I and instructionsfor using the compound to (a) suppress an immune response in a patient,(b) treat or prevent an immune-mediated disease or condition in apatient, or (c) lower the toxicity profile of a second drug. In someembodiments, the immune response is inflammation. In some embodimentsthe immune response is transplant rejection. In some embodiments, theimmune-mediated disease or condition is selected from the groupconsisting of auto-immune diseases, inflammation processes, transplantrejection, allergic reactions. In some embodiments, the immune-mediateddisease or condition is selected from Psoriasis, lichen planus and otherpapulosquamous disorders. In some embodi-ments, the immune-mediateddisease or condition is selected from eczema and dermatitis. In someembodiments, the eczemea or dermatitis is selected from eczema, atopiceczema, sebor-rheic dermatitis, and pompholyx. In some embodiments, theimmune-mediated disease or con-dition is a skin reaction to sunlight. Insome embodiments, the immune-mediated disease or condition selected fromnon-specific skin irritation and insect bite. In some embodiments, theimmune-mediated disease or condition is a urticaria. In someembodiments, the immune-mediated disease or condition is selected fromthe group consisting of a primary skin tumor (e.g. melanoma); rheumatoidarthritis (both autoimmune and elicited by infection); Crohn's disease;inflammatory bowel disease; irritable bowel syndrome; aneurodegenerative disease (e.g. multiple sclerosis); Parkinson'sdisease; Graft-versus-Host reaction; severe psoriasis; and atopicdermatitis. In some embodiments, the instructions instruct administeringthe compound of formula I in conjunction with a chemotherapeutic drug inorder to reduce the toxic effects of the chemotherapeutic drug. In someembodiments, the compound is a compound of formula I-1. In someembodiments, the compound is a compound of formula I-2. In someembodiments, the compound is a compound of formula I-3. In someembodiments, the compound is a compound of formula I-4. In someembodiments, the compound is a compound of formula I-5. In someembodiments, the compound is a compound of formula I-6. In someembodiments, the compound is a compound of formula I-7. In someembodiments, the compound is a compound of formula I-8. In someembodiments, the compound is a compound of formula I-A. In someembodiments, the compound is a compound of formula I-B. In someembodiments, the compound is a compound of formula I-C. In someembodiments, the compound is a compound of formula I-D. In someembodiments, the compound is a compound of formula I-E. In someembodiments, the compound is a compound of formula I-F. In someembodiments, the compound is a compound of formula I-G. In someembodiments, the compound is a compound of formula I-H. In someembodiments, the compound is a compound of formula I-I. In someembodiments, the compound is a compound of formula I-J. In someembodiments, the compound is a compound of formula I-K. In someembodiments, the compound is a compound of formula I-L. In someembodiments, the compound is a compound of formula I-M. In someembodiments, the compound is a compound of formula I-N. In someembodiments, the compound is a compound of formula I-O. In someembodiments, the compound is a compound of formula I-P.

There is also provided, in accordance with an embodiment of theinvention, a method for making a compound of formula I, comprisingcyclizing a compound having the formula II-1, II-2, II-3 or II-4,wherein R, R′, R″, R′″, R³, R⁴, k, m, n and p are as defined in formulaI (collectively referred to hereinafter as compounds of formula II):

to the corresponding compound of formula I. In some embodiments, themethod further comprises synthesizing the compound of formula II-1,II-2, II-3 or II-4. In some embodiments, the compound of formula II-1,II-2, II-3 or II-4 is formed by solid-phase synthesis.

In accordance with embodiments of the invention, there are also providedcompounds of formulae II-1, II-2, II-3 and II-4 per se, as well asprotected versions of these compounds (e.g. in which one or more aminogroups, such as the N-terminal amino group or a side chain amino group,are protected, e.g. by tert-butoxycarbonyl) and these compounds, inprotected or unprotected form, when bound to a solid-phase resin.Hereinafter, unless specified otherwise or illogical in the givencontext, when reference is made to a compound of formula II or asub-genus or sub-species thereof, such reference is intended to includesuch compound in a form in which it is (a)(i) at least partly protectedor (a)(ii) completely un- or deprotected; (b)(i) bound to a resin (1)directly or (2) through a linker, or (b)(ii) not bound to a resin; or acombination of conditions (a) and (b). Furthermore, as depicted herein,for the sake of convenience, free, non-protected linear peptides areshown as neutral molecules, viz. having H₂N— at the N-terminus and —COOHat the C-terminus; however, it will be appreciated that the actualcharge on these moieties, as well as on any ionizable side chainmoieties (e.g. carboxylic acid or amine moieties in the side chains)will depend on the pH of surroundings, and will not necessarily be asshown.

In some embodiments, the compound of formula II is selected from thegroup consisting of:

In some embodiments the compound is a compound of formula II-4-a. Insome embodiments, the compound is a compound of formula II-1-a. In someembodiments, the compound is a compound of formula II-2-a. In someembodiments, the compound is a compound of formula II-3-a. In someembodiments, the compound is a compound of formula II-4-b. In someembodiments, the compound is a compound of formula II-1-b. In someembodiments, the compound is a compound of formula II-2-b. In someembodiments, the compound is a compound of formula II-3-b. In someembodiments, the compound is a compound of formula II-4-c. In someembodiments, the compound is a compound of formula II-1-c. In someembodiments, the compound is a compound of formula II-2-c. In someembodiments, the compound is a compound of formula II-3-c. In someembodiments, the compound is a compound of formula II-4-d. In someembodiments, the compound is a compound of formula II-1-d. In someembodiments, the compound is a compound of formula II-2-d. In someembodiments, the compound is a compound of formula II-3-d. In someembodiments, the compound is a compound of formula II-4-e. In someembodiments, the compound is a compound of formula II-1-e. In someembodiments, the compound is a compound of formula II-2-e. In someembodiments, the compound is a compound of formula II-3-e. In someembodiments, the compound is a compound of formula II-4-f. In someembodiments, the compound is a compound of formula II-1-f. In someembodiments, the compound is a compound of formula II-2-f. In someembodiments, the compound is a compound of formula II-3-f. In someembodiments, the compound is a compound of formula II-4-g. In someembodiments, the compound is compound of formula II-1-g. In someembodiments, the compound is compound of formula II-2-g. In someembodiments, the compound is compound of formula II-3-g. In someembodiments, the compound is a compound of formula II-4-h. In someembodiments, the compound is compound of formula II-1-h. In someembodiments, the compound is compound of formula II-2-h. In someembodiments, the compound is compound of formula II-3-h.

In some embodiments the compound of formula II is selected from thegroup consisting of:

In some embodiments, the compound is a compound of formula II-A-1. Insome embodiments, the compound is a compound of formula II-A-2. In someembodiments, the compound is a compound of formula II-A-3. In someembodiments, the compound is a compound of formula II-A-4. In someembodiments, the compound is a compound of formula II-B-1. In someembodiments, the compound is a compound of formula II-B-2. In someembodi-ments, the compound is a compound of formula II-B-3. In someembodiments, the compound is a compound of formula II-B-4. In someembodiments, the compound is a compound of formula II-C-1. In someembodiments, the compound is a compound of formula II-C-2. In someembodiments, the compound is a compound of formula II-C-3. In someembodiments, the compound is a compound of formula II-C-4. In someembodiments, the compound is a compound of formula II-D-1. In someembodiments, the compound is a compound of formula II-D-2. In someembodiments, the compound is a compound of formula II-D-3. In someembodiments, the compound is a compound of formula II-D-4. In someembodiments, the compound is a compound of formula II-E-1. In someembodiments, the compound is a compound of formula II-E-2. In someembodiments, the compound is a compound of formula II-E-3. In someembodiments, the compound is a compound of formula II-E-4. In someembodiments, the compound is a compound of formula II-F-1. In someembodiments, the compound is a compound of formula II-F-2. In someembodiments, the compound is a compound of formula II-F-3. In someembodiments, the compound is a compound of formula II-F-4. In someembodiments, the compound is a compound of formula II-G-1. In someembodiments, the compound is a compound of formula II-G-2. In someembodiments, the compound is a compound of formula II-G-3. In someembodiments, the compound is a compound of formula II-G-4. In someembodiments, the compound is a compound of formula II-H-1. In someembodiments, the compound is a compound of formula II-H-2. In someembodiments, the compound is a compound of formula II-H-3. In someembodiments, the compound is a compound of formula II-H-4. In someembodiments, the compound is a compound of formula II-J-1. In someembodiments, the compound is a compound of formula II-J-2. In someembodiments, the compound is a compound of formula II-J-3. In someembodiments, the compound is a compound of formula II-J-4. In someembodiments, the compound is a compound of formula II-K-1. In someembodiments, the compound is a compound of formula II-K-2. In someembodiments, the compound is a compound of formula II-K-3. In someembodiments, the compound is a compound of formula II-K-4. In someembodiments, the compound is a compound of formula II-L-1. In someembodiments, the compound is a compound of formula II-L-2. In someembodiments, the compound is a compound of formula II-L-3. In someembodiments, the compound is a compound of formula II-L-4. In someembodiments, the compound is a compound of formula II-M-1. In someembodiments, the compound is a compound of formula II-M-2. In someembodiments, the compound is a compound of formula II-M-3. In someembodiments, the compound is a compound of formula II-M-4. In someembodiments, the compound is a compound of formula II-N-1. In someembodiments, the compound is a compound of formula II-N-2. In someembodiments, the compound is a compound of formula II-N-3. In someembodiments, the compound is a compound of formula II-N-4. In someembodiments, the compound is a compound of formula II-O-1. In someembodiments, the compound is a compound of formula II-O-2. In someembodiments, the compound is a compound of formula II-O-3. In someembodiments, the compound is a compound of formula II-O-4. In someembodiments, the compound is a compound of formula II-P-1. In someembodiments, the compound is a compound of formula II-P-2. In someembodiments, the compound is a compound of formula II-P-3. In someembodiments, the compound is a compound of formula II-P-4.

DETAILED DESCRIPTION

It has been found that compounds of formula I exhibit immunosuppressiveand/or anti-inflammatory activity, while at the same time exhibitingless toxicity, than some known compounds. Thus compounds of formula Imay be useful as immunosuppressive and/or anti-inflammatory agents. Asused herein, the term “immune-mediated” refers to a disease or conditionin which the body's immune system overreacts and/or attacks the body.

Throughout this specification the terms and substituents retain theirdefinitions.

“Alkyl” is intended to include linear, branched, or cyclic saturatedhydrocarbon structures and combinations thereof. Lower alkyl refers toalkyl groups of from 1 to 6 carbon atoms. Examples of lower alkyl groupsinclude methyl, ethyl, propyl, isopropyl, butyl, s- and t-butyl and thelike. Preferred alkyl groups are those of C₂₀ or below. Cycloalkyl is asubset of alkyl and includes cyclic hydrocarbon groups of from 3 to 8carbon atoms. Examples of cycloalkyl groups include c-propyl, c-butyl,c-pentyl, norbornyl and the like.

C₁ to C₂₀ hydrocarbon includes alkyl, cycloalkyl, polycycloalkyl,alkenyl, alkynyl, aryl and combinations thereof. Examples includebenzyl, phenethyl, cyclohexylmethyl, camphoryl and naphthylethyl. Theterm “carbocycle” is intended to include ring systems consistingentirely of carbon but of any oxidation state. Thus (C₃-C₁₀) carbocyclerefers to such systems as cyclopropane, benzene and cyclohexene;(C₈-C₁₂) carbopolycycle refers to such systems as norbornane, decalin,indane and naphthalene.

Alkoxy or alkoxyl refers to groups of from 1 to 8 carbon atoms of astraight, branched, cyclic configuration and combinations thereofattached to the parent structure through an oxygen atom. Examplesinclude methoxy, ethoxy, propoxy, isopropoxy, cyclopropyloxy,cyclohexyloxy and the like. Lower-alkoxy refers to groups containing oneto four carbons.

Oxaalkyl refers to alkyl residues in which one or more carbons has beenreplaced by oxygen. Examples include methoxypropoxy, 3,6,9-trioxadecyland the like.

Acyl refers to groups of from 1 to 8 carbon atoms of a straight,branched, cyclic configuration, saturated, unsaturated and aromatic andcombinations thereof, attached to the parent structure through acarbonyl functionality. One or more carbons in the acyl residue may bereplaced by nitrogen, oxygen or sulfur as long as the point ofattachment to the parent remains at the carbonyl. Examples includeacetyl, benzoyl, propionyl, isobutyryl, t-butoxycarbonyl,benzyloxycarbonyl and the like. Lower-acyl refers to groups containingone to four carbons.

Aryl means 6-membered aromatic ring; a bicyclic 9- or 10-memberedaromatic ring system; or a tricyclic 13- or 14-membered aromatic ringsystem. The aromatic 6- to 14-membered carbocyclic rings include, e.g.,benzene, naphthalene, indane, tetralin, and fluorene.

Heteroaryl mean a 5- or 6-membered heteroaromatic ring containing 1-3heteroatoms selected from O, N, or S; a bicyclic 9- or 10-memberedheteroaromatic ring system containing 1-3 heteroatoms selected from O,N, or S; or a tricyclic 13- or 14-membered heteroaromatic ring systemcontaining 1-3 heteroatoms selected from O, N, or S. The 5- to10-membered aromatic heterocyclic rings include, e.g., imidazole,pyridine, indole, thiophene, benzopyranone, thiazole, furan,benzimidazole, quinoline, isoquinoline, quinoxaline, pyrimidine,pyrazine, tetrazole and pyrazole.

Arylalkyl refers to a substituent in which an aryl residue is attachedto the parent structure through alkyl. Examples are benzyl, phenethyland the like. Heteroarylalkyl refers to a substituent in which aheteroaryl residue is attached to the parent structure through alkyl.Examples include, e.g., pyridinylmethyl, pyrimidinylethyl and the like.

Heterocycle means a cycloalkyl or aryl residue in which from one tothree carbons is replaced by a heteroatom selected from the groupconsisting of N, O and S. The nitrogen and sulfur heteroatoms mayoptionally be oxidized, and the nitrogen heteroatom may optionally bequaternized. Examples of heterocycles include pyrrolidine, pyrazole,pyrrole, indole, quinoline, isoquinoline, tetrahydroisoquinoline,benzofuran, benzodioxan, benzodioxole (commonly referred to asmethylenedioxyphenyl, when occurring as a substituent), tetrazole,morpholine, thiazole, pyridine, pyridazine, pyrimidine, thiophene,furan, oxazole, oxazoline, isoxazole, dioxane, tetrahydrofuran and thelike. It is to be noted that heteroaryl is a subset of heterocycle inwhich the heterocycle is aromatic. Examples of heterocyclyl residuesadditionally include piperazinyl, 2-oxopiperazinyl, 2-oxopiperidinyl,2-oxo-pyrrolidinyl, 2-oxoazepinyl, azepinyl, 4-piperidinyl,pyrazolidinyl, imidazolyl, imidazolinyl, imidazolidinyl, pyrazinyl,oxazolidinyl, isoxazolidinyl, thiazolidinyl, isothiazolyl,quinuclidinyl, isothiazolidinyl, benzimidazolyl, thiadiazolyl,benzopyranyl, benzothiazolyl, tetrahydrofuryl, tetrahydropyranyl,thienyl, benzothienyl, thiamorpholinyl, thiamorpholinylsulfoxide,thiamorpholinylsulfone, oxadiazolyl, triazolyl and tetrahydroquinolinyl.

Substituted alkyl, aryl, cycloalkyl, heterocyclyl etc. refer to alkyl,aryl, cycloalkyl, or heterocyclyl wherein up to three H atoms in eachresidue are replaced with alkyl, halogen, haloalkyl, hydroxy,loweralkoxy, carboxy, carboalkoxy (also referred to as alkoxycarbonyl),carboxamido (also referred to as alkylaminocarbonyl), cyano, carbonyl,nitro, amino, alkylamino, dialkylamino, mercapto, alkylthio, sulfoxide,sulfone, acylamino, amidino, phenyl, benzyl, heteroaryl, phenoxy,benzyloxy, or heteroaryloxy.

The term “halogen” means fluorine, chlorine, bromine or iodine.

The following abbreviations and terms have the indicated meaningsthroughout:

-   -   Boc=t-butyloxy carbonyl    -   c-=cyclo    -   DCM=dichloromethane=methylene chloride=CH₂Cl₂    -   DIEA=N,N-diisopropylethyl amine    -   DIPEA=diisopropylethylamine    -   DMF=N,N-dimethylformamide    -   Fmoc=9-fluorenylmethoxycarbonyl    -   HATU=O-(7-Azabenzotriazol-1-yl)-1,1,3,3-tetramethyluronium        hexafluorophosphate    -   HBTU=O-(benzotriazol-1-yl)-1,1,3,3-tetramethyluronium        hexafluorophosphate    -   HOAc=acetic acid    -   HOAt=1-hydroxy-7-azabenzotriazole    -   HOBt=1-hydroxybenzotriazole    -   Hyp=4-hydroxyproline    -   Me=methyl    -   Pip=Pipecolic acid    -   Phe=phenylalanine    -   Pro=proline    -   PyBOP=O-(benzotriazol-1-yl)-trispyrrolidinephosphonium        hexafluorophosphate    -   rt=room temperature    -   TBTU=O-(benzotriazol-1-yl)-1,1,3,3-tetramethyluronium        tetrafluoroborate    -   TFA=trifluoroacetic acid    -   t-Hyp=trans-4-hydroxyproline    -   Trp=tryptophan    -   Tyr=tyrosine    -   Tyr(tBu)=(O-tert-butyl)tyrosine

Furthermore, a comprehensive list of abbreviations utilized by organicchemists (i.e. persons of ordinary skill in the art) appears in thefirst issue of each volume of the Journal of Organic Chemistry. Thelist, which is typically presented in a table entitled “Standard List ofAbbreviations” is incorporated herein by reference. In addition, withrespect to non-naturally occurring amino acids in which an additionalmethylene group is present in the backbone, the notation “β³-Ho-” (or“beta 3-homo-”) is used herein to refer to an amino acid having an extramethylene (—CH₂—) in the backbone between the side-chain bearing carbonatom and the terminal nitrogen atom; the notation “β²-Ho-” (or “beta2-homo-”) is used herein to refer to an amino acid having an extramethylene in the backbone between the side-chain bearing carbon atom andthe terminal carbon atom.

Embodiments of the present invention include compounds of formula I inthe form of salts, in particular acid addition salts. Suitable saltsinclude those formed with both organic and inorganic acids. Such acidaddition salts will normally be pharmaceutically acceptable, althoughsalts of non-pharmaceutically acceptable salts may be of utility in thepreparation and purification of the compound in question. Thus,preferred salts include those formed from hydrochloric, hydrobromic,sulphuric, citric, tartaric, phosphoric, lactic, pyruvic, acetic,succinic, oxalic, fumaric, maleic, oxaloacetic, methanesulphonic,ethanesulphonic, p-toluenesulphonic, benzenesulphonic and isethionicacids. Salts of the compounds of formula I can be made by reacting theappropriate compound in the form of the free base with the appropriateacid.

The compounds of formula I in accordance with the embodiments of theinvention are cyclic tetrapeptides. The synthesis of these peptides maybe accomplished by cyclizing corresponding linear peptides that arethemselves synthesized using methodologies known in the art; see, forexample, Merrifield, J. Am. Chem. Soc., 85:2149 (1964); Houghten, Proc.Natl. Acad. Sci. USA, 82:5132 (1985); Kelley & Winkler in GeneticEngineering Principles and Methods, Setlow, J. K, ed., Plenum Press,N.Y., vol. 12, pp 1-19 (1990); Stewart & Young, Solid Phase PeptideSynthesis, Pierce Chemical Co., Rockford, Ill. (1984); Mergler et al.(1988) Tetrahedron Letters 29:4005-4008; Mergler et al. (1988)Tetrahedron Letters 29:4009-4012; Kamber et al. (eds), Peptides,Chemistry and Biology, ESCOM, Leiden (1992) pp. 525-526; Riniker et al.(1993) Tetrahedron Letters 49:9307-9320; Lloyd-Williams et al. (1993)Tetrahedron Letters 49:11065-11133; Andersson et al. (2000) Biopolymers55:227-250; Bray, Nature Reviews 2:587-593 (2003), U.S. Pat. Nos.4,105,603, 3,972,859, 3,842,067, 3,862,925, 6,015,881, 6,197,927, and7,439,222. Such synthesis may be accomplish via liquid-phase orsolid-phase synthesis, or by a combination of both, as is known in theart.

Liquid phase methods (often referred to as solution phase methods) ofsynthesis carry out all reactions in a homogeneous phase. Successiveamino acids are coupled in solution until the desired peptide materialis formed. During synthesis, successive intermediate peptides arepurified by precipitation and/or washes.

In solid phase peptide synthesis (SPPS), a first amino acid or peptidegroup is bound to an insoluble support, such as a resin. Successiveamino acids or peptide groups are added to the first amino acid orpeptide group until the peptide material of interest is formed. Theproduct of solid phase synthesis is thus a peptide bound to an insolublesupport. Peptides synthesized via SPPS techniques are then cleaved fromthe resin, and the cleaved peptide is isolated.

More specifically, solid phase synthesis begins at the carboxy terminusof the putative peptide by coupling a protected amino acid to an inertsolid support. The inert solid support can be any macromolecule capableof serving as an anchor for the C-terminus of the initial amino acid.Typically, the macromolecular support is a cross-linked polymeric resin(e.g. a polyamide or polystyrene resin) as shown in FIGS. 1-1 and 1-2,on pages 2 and 4 of Stewart & Young, supra. In some cases, theC-terminal amino acid is coupled to a polystyrene resin to form a benzylester. A macromolecular support is selected such that the peptide anchorlink is stable under the conditions used to deprotect the α-amino groupof the blocked amino acids in peptide synthesis. If a base-labileα-protecting group is used, then it is desirable to use an acid-labilelink between the peptide and the solid support. For example, anacid-labile ether resin is effective for base-labile Fmoc-amino acidpeptide synthesis as described on page 16 of Stewart & Young, supra.Alternatively, a peptide anchor link and α-protecting group that aredifferentially labile to acidolysis can be used. For example, anaminomethyl resin such as the phenylacetamidomethyl (Pam) resin workswell in conjunction with Boc-amino acid peptide synthesis as describedon pages 11-12 of Stewart & Young, supra. Guiller et al., Chem. Rev.2000, 100, 2091-2157, reviewed linkers and cleavage strategies insolid-phase organic synthesis and combinatorial chemistry, includingpeptide synthesis.

After the initial amino acid is coupled to an inert solid support, theα-amino protecting group of the initial amino acid is removed with, forexample, trifluoroacetic acid (TFA) in methylene chloride andneutralizing in, for example, triethylamine (TEA). Followingdeprotection of the initial amino acid's α-amino group, the next α-aminoand sidechain protected amino acid in the synthesis is added. Theremaining α-amino and, if necessary, side chain protected amino acidsare then coupled sequentially in the desired order by condensation toobtain an intermediate compound connected to the solid support.Alternatively, some amino acids may be coupled to one another to form afragment of the desired peptide followed by addition of the peptidefragment to the growing solid phase peptide chain

The condensation reaction between two amino acids, or an amino acid anda peptide, or a peptide and a peptide can be carried out according tothe usual condensation methods such as the axide method, mixed acidanhydride method, DCC(N,N′-dicyclohexylcarbodiimide) orDIC(N,N′-diisopropylcarbodiimide) methods, active ester method,p-nitrophenyl ester method, BOP (benzotriazole-1-yl-oxy-tris[dimethylamino]phosphonium hexafluorophosphate) method,N-hydroxysuccinic acid imido ester method, etc, and Woodward reagent Kmethod.

It is common in the chemical syntheses of peptides to protect anyreactive side-chain groups of the amino acids with suitable protectinggroups. Ultimately, these protecting groups are removed after thedesired polypeptide chain has been sequentially assembled. Also commonis the protection of the α-amino group on an amino acid or peptidefragment while the C-terminal carboxy group of the amino acid or peptidefragment reacts with the free N-terminal amino group of the growingsolid phase polypeptide chain, followed by the selective removal of theα-amino protecting group to permit the addition of the next amino acidor peptide fragment to the solid phase polypeptide chain. Accordingly,it is common in polypeptide synthesis that an intermediate compound isproduced which contains each of the amino acid residues located in thedesired sequence in the peptide chain wherein individual residues stillcarry side-chain protecting groups. These protecting groups can beremoved substantially at the same time to produce the desiredpolypeptide product following removal from the solid phase.

α- and ω-amino side chains can be protected, for example, withbenzyloxycarbonyl (abbreviated Z), isonicotinyloxycarbonyl (iNoc),o-chlorobenzyloxycarbonyl [Z(2Cl) or 2-Cl—Z], p-nitrobenzyloxycarbonyl[Z(NO₂)], p-methoxybenzyloxycarbonyl [Z(OMe)], t-butoxy-carbonyl (Boc),t-amyloxycarbonyl (Aoc), isobornyloxycarbonyl, adamantyloxy-carbonyl(Adoc), 2-(4-biphenyl)-2-propyloxycarbonyl (Bpoc),9-fluorenylmethoxycarbonyl (Fmoc), methylsulfonyethoxycarbonyl (Msc),trifluoroacetyl, phthalyl (Pht), formyl (For), 2-nitro-phenylsulphenyl(Nps), diphenylphosphinothioyl (Ppt), and dimethylphosphinothioyl (Mpt)groups, and the like. Additional examples of side chain protectinggroups include acetyl (Ac), benzoyl (Bz), tert butyl (t-Bu),triphenylmethyl (trityl, Trt), tetrahydropyranyl, benzyl (Bzl),2,6-dichlorobenzyl, nitro, p-toluenesulfonyl (Tos), xanthyl (Xan),benzyl, methyl, ethyl, and t-butyl ester, and aromatic or aliphaticurethan-type protecting groups, photolabile groups such as nitroveratryl oxycarbonyl (Nvoc), and fluoride labile groups such astrimethylsilylethyloxycarbonyl (TEOC).

Examples of amino terminal protecting groups (also referred to herein asN-terminal protecting groups) include: (1) acyl-type protecting groups,such as formyl, acrylyl (Acr), benzoyl (Bz) and acetyl (Ac); (2)aromatic urethan-type protecting groups, such as benzyloxy-carbonyl (Z)and substituted Z, such as p-chlorobenzyloxycarbonyl,p-nitrobenzyloxycarbonyl, p-bromobenzyloxycarbonyl,p-methoxybenzyloxycarbonyl; (3) aliphatic urethan protecting groups,such as t-butyloxycarbonyl (BOC), diisopropylmethoxycarbonyl,isopropyloxycar-bonyl, ethoxycarbonyl, allyloxycarbonyl; (4) cycloalkylurethan-type protecting groups, such as 9-fluorenyl-methyloxycarbonyl(Fmoc), cyclopentyloxycarbonyl, adamantyloxycarbonyl, andcyclohexyloxycarbonyl; and (5) thiourethan-type protecting groups, suchas phenylthio-carbonyl. Preferred protecting groups include9-fluorenylmethyloxycarbonyl (Fmoc),2-(4-biphenylyl)-propyl(2)oxycarbonyl (Bpoc),2-phyenlpropyl(2)-oxycarbonyl (Poc), and t-butyloxycarbonyl (Boc).

Protective groups for the carboxy functional group are exemplified bybenzyl ester (OBzl), cyclohexyl ester (Chx), 4-nitrobenzyl ester (ONb),t-butyl ester (Obut), 4-pyridylmeth-yl ester (OPic), and the like. It isoften desirable that specific amino acids such as arginine, cysteine,and serine possessing a functional group other than amino and carboxylgroups are protected by a suitable protective group. For example, theguanidino group of arginine may be protected with nitro,p-toluenesulfonyl, benzyloxycarbonyl, adamantyloxycarbonyl,p-meth-oxybenzesulfonyl, 4-methoxy-2,6-dimethylbenzenesulfonyl (Nds),1,3,5-trimethylphenysul-fonyl (Mts), and the like. The thiol group ofcysteine can be protected with p-methoxybenzyl, trityl, and the like.

While it may be possible for the compounds of formula Ito beadministered as the raw chemical, it is preferable to present them as apharmaceutical composition. According to a further aspect, there areprovided in accordance with embodiments of the present invention apharmaceutical composition comprising a compound of formula I or apharmaceutically acceptable salt or solvate thereof, together with oneor more pharmaceutically carriers thereof and optionally one or moreother therapeutic ingredients. The carrier(s) must be “acceptable” inthe sense of being compatible with the other ingredients of theformulation and not deleterious to the recipient thereof.

The formulations include those suitable for oral, parenteral (includingsubcutaneous, intradermal, intramuscular, intravenous andintraarticular), rectal and topical (including dermal, buccal,sublingual and intraocular) administration. The most suitable route maydepend upon the condition and disorder of the recipient. Theformulations may conveniently be presented in unit dosage form and maybe prepared by any of the methods well known in the art of pharmacy.Methods for making such formulations include the step of bringing intoassociation a compound of formula I or a pharmaceutically acceptablesalt or solvate thereof (“active ingredient”) with the carrier whichconstitutes one or more accessory ingredients. In general, theformulations are prepared by uniformly and intimately bringing intoassociation the active ingredient with liquid carriers or finely dividedsolid carriers or both and then, if necessary, shaping the product intothe desired formulation.

Formulations in accordance with embodiments of the present inventionsuitable for oral administration may be presented as discrete units suchas capsules, cachets or tablets each containing a predetermined amountof the active ingredient; as a powder or granules; as a solution or asuspension in an aqueous liquid or a non-aqueous liquid; or as anoil-in-water liquid emulsion or a water-in-oil liquid emulsion. Theactive ingredient may also be presented as a bolus, electuary or paste.

A tablet may be made by compression or molding, optionally with one ormore accessory ingredients. Compressed tablets may be prepared bycompressing in a suitable machine the active ingredient in afree-flowing form such as a powder or granules, optionally mixed with abinder, lubricant, inert diluent, lubricating, surface active ordispersing agent. Molded tablets may be made by molding in a suitablemachine a mixture of the powdered compound moistened with an inertliquid diluent. The tablets may optionally be coated or scored and maybe formulated so as to provide sustained, delayed or controlled releaseof the active ingredient therein.

Formulations for parenteral administration include aqueous andnon-aqueous sterile injection solutions which may contain anti-oxidants,buffers, bacteriostats and solutes which render the formulation isotonicwith the blood of the intended recipient. Formulations for parenteraladministration also include aqueous and non-aqueous sterile suspensions,which may include suspending agents and thickening agents. Theformulations may be presented in unit-dose of multi-dose containers, forexample sealed ampoules and vials, and may be stored in a freeze-dried(lyophilized) condition requiring only the addition of a sterile liquidcarrier, for example saline, phosphate-buffered saline (PBS) or thelike, immediately prior to use. Extemporaneous injection solutions andsuspensions may be prepared from sterile powders, granules and tabletsof the kind previously described.

Formulations for rectal administration may be presented as a suppositorywith the usual carriers such as cocoa butter or polyethylene glycol.

Formulations for topical administration in the mouth, for examplebuccally or sublingually, include lozenges comprising the activeingredient in a flavoured basis such as sucrose and acacia ortragacanth, and pastilles comprising the active ingredient in a basissuch as gelatin and glycerin or sucrose and acacia.

Preferred unit dosage formulations are those containing an effectivedose, as hereinbelow recited, or an appropriate fraction thereof, of theactive ingredient.

It should be understood that in addition to the ingredients particularlymentioned above, the formulations in accordance with embodiments of thisinvention may include other agents conventional in the art having regardto the type of formulation in question, for example those suitable fororal administration may include flavoring agents.

As stated, in accordance with embodiments of the invention, compounds inaccordance with embodiments of the invention may be used for thetreatment or prevention of certain diseases or conditions. The term“preventing” as used herein refers to administering a medicamentbeforehand to forestall or obtund an attack. The person of ordinaryskill in the medical art (to which the present method of use claims aredirected) recognizes that the term “prevent” is not an absolute term. Inthe medical art it is understood to refer to the prophylacticadministration of a drug to substantially diminish the likelihood orseriousness of a condition, and this is the sense intended inapplicants' claims. The reader's attention is directed to thePhysician's Desk Reference, a standard text in the field, in which theterm “prevent” occurs hundreds of times. No person of skill in themedical art construes the term in an absolute sense. Similarly, where itis stated that compounds in accordance with embodiments of the inventionmay be used to suppress an immune response, “suppress” will beunderstood to include reducing the degree of the response, and notnecessarily absolutely preventing the response.

It may be found upon examination that compounds that are not presentlyexcluded from the claims are not patentable to the inventors in thisapplication. In that case, the exclusion of species and genera inapplicants' claims are to be considered artifacts of patent prosecutionand not reflective of the inventors' concept or description of theirinvention. The invention, in a composition aspect, is all compounds offormulae I and II, except those that are in the public's possession.

Embodiments of the invention will be better understood with reference tothe figures, in which:

FIG. 1 shows antigen-specific increase of the ear thickness derived fromthe experiment described in Example 2;

FIG. 2 shows total numbers of cells in the parotid lymph nodes observedin the experiment described in Example 2;

FIG. 3 shows the content of viable and dead cells in the parotid lymphnodes described in Example 2;

FIG. 4 presents number and participation of cell types in the draininglymph nodes registered in the experiment described in Example 2;

FIG. 5 presents toxicity of the compound of formula IA againstmononuclear cells from human blood in as described in Example 3;

FIG. 6 presents effects of the intraperitoneal administration of thecompound of formula IA on the humoral immune response of mice to sheeperythrocytes as described in Example 4;

FIG. 7 shows effects of the intraperitoneal administration of thecompound of formula IA on the cellular immune response to ovalbumin asdescribed in Example 5;

FIGS. 8A and 8B show the effects of peptides in accordance withembodiments of the invention on PMBC survival in vitro;

FIGS. 9A and 9B show the effects of peptides in accordance withembodiments of the invention on PHA-induced PBMC proliferation in vitro;

FIGS. 10A and 10B show the effects of several compounds on changes inear thickness in response to antigenic challenge, as described inExample 7;

FIG. 11 shows the permeability of capillary blood vessels in the Evansblue test, as described in Example 7;

FIG. 12 shows the total number of cells in the draining lymph nodes, asdescribed in Example 7;

FIG. 13 shows the effects of the compounds on the numbers of circulatingleukocytes, as described in Example 7;

FIG. 14 which shows the types of leukocytes present in different cases,as described in Example 7; and

FIG. 15 provides morphometric data on the number and composition ofcells in mouse auricles, as described in Example 7.

Example 1 Synthesis of Cyclic Tetrapeptides

Cyclic tetrapeptides according to embodiments of the invention can besynthesized by the use of known peptide synthesis methods, such assolution-phase or solid-phase methods. In general the synthesis involvestwo consecutive steps: (1) synthesis of a linear tetrapeptide and (2)cyclization to yield the cyclic tetrapeptide. The linear tetrapeptidemay be prepared in protected form and then deprotected prior tocyclization.

To illustrate, the synthesis of linear tetrapeptides on a solid support(Fmoc-L-phenylalanine attached to a Wang-type resin, or eitherL-beta-3-homophenylalanine or L-proline attached to a 2-chloro tritylresin) was conducted according to the following protocol:

1. The resin was swelled in dimethylformamide (DMF) (0.25 mmol, 10 ml/gresin) for 15 min.2. The Fmoc group was removed with a 20% solution of piperidine in DMF(2×20 min).3. The resin was washed with DMF (3×2 min)4. The resin was washed with methanol (MeOH) (3×2 min)5. The resin was washed with dichloromethane (DCM) (3×2 min)6. Amino acid or peptide amino groups on the resin were acylated byshaking with a mixture of Fmoc-protected amino acid (4 eq), HBTU or TBTU(4 eq) and DIPEA (4 eq) dissolved in anhydrous DMF (4 ml/mmol), for 20hours.7. The resin was washed with DCM (3×2 min)8. The resin was washed with MeOH (3×2 min)9. The resin was washed with DCM (3×2 min)10. The Kaiser test (for all amino acids except proline) was used toestimate if all amino groups were acylated.

If the result of the Kaiser test was negative, the resin was washed withDMF (1×2 min.) and a new coupling cycle started from point 2 of theprotocol. If the result of the Kaiser test was positive, acylation wasrepeated with half the amount of the reagent used for the firstcoupling. In the case of acylation of proline, the Kaiser test is notsensitive enough to determine the degree of acylation, and for thatreason in the case of proline the coupling procedure was repeated withthe half amount of reagents. In the case of repeated acylation, thefollowing washings were carried out:

7A. Washing with DCM (3×2 min)8A. Washing with MeOH (3×2 min)9A. Washing with DCM (3×2 min)

After the last coupling the resin was washed according to points 3-5 ofthe protocol. The Fmoc group was removed from the peptide as describedin point 2, and the resin was washed again with DMF, MeOH and DCM as inpoints 3-5. Before the cleavage of the peptide from the polymericsupport, the resin was dried overnight in a desiccator over KOH pelletsunder reduced pressure at room temperature.

Peptides were cleaved from the dried, Wang-type resin with a mixture oftrifluoroacetic acid/water/triisopropylsilane 95:2.5:2.5 (v/v/v; 10 ml/1g of peptidyl-resin). The solution obtained was partially evaporated atroom temperature under reduced pressure and peptide was precipitatedwith 10 volumes of ether. After being filtered off, the crude peptidewas dissolved in 0.05 M aqueous HCl and evaporated to dryness. Theresidue was dissolved in water and lyophilized.

Peptides were cleaved from 2-Cl-trityl resin by treatment with a 1:3:1mixture of acetic acid/dichloromethane/trifluorethanol (v/v/v; 10 ml/1 gof peptidyl-resin). The resulting cleavage solution plus collectedwashings were filtered, evaporated to dryness at ambient temperature andreduced pressure, and the residue dissolved in a minimal volume of DCM,diluted with 20 volumes of hexane and re-evaporated (twice). Crudedetached peptide was dissolved in water and lyophilized.

After lyophilized peptide was dried in a desiccator under vacuum overKOH and P₂O₅, it was ready for cyclisation.

In a typical synthesis following the above protocol, starting withFmoc-Phe attached to the Wang resin (278 mg, 0.2 mmol, 0.72 mmol/g) andafter sequential coupling of Fmoc-L-β³-homoPhe-OH, Fmoc-Pro-OH and againFmoc-Pro-OH, the yield after lyophylization was 91 mg (82%) of crudelinear peptide with a purity of 94% (HPLC).

Crude peptides were cyclized in DCM solution (peptide concentration2×10⁻⁴ millimoles/Liter) with the aid of PyBOP/HOAt/2,4,6-collidine(3:3:5), with disappearance of linear precursors being traced byinjecting samples of reaction solutions on an analytical reverse-phase(RP)HPLC column. At the end of the cyclization, the solution wasevaporated to dryness under reduced pressure and the residue waspartitioned between ethyl acetate (1000 ml of solvent per 1 millimole ofpeptide and 0.5 N HCl in water (100 ml of solution per 1 millimole ofpeptide). Organic phase was washed consecutively with 0.5 N HCl in water(2×), water (1×), 1M NaHCO₃ (3×) and water (1×). Organic solvent wasremoved under reduced pressure and the residual solid was dissolved indioxane and freeze-dried. The crude product was purified on apreparative Vydac C₁₈ or Kromasil C₈ reversed-phase column (250 mm/22mm, 100 A, 10 μm) using an elution gradient of solvent B (0.038% TFA in82% acetonitrile/water) in solvent A (0.05% TFA in water).

As an example of a cyclization reaction, a solution ofH-Pro-Pro-β³-hPhe-Phe-OH×HCl (55.7 mg, 0.1 mmol) in 500 ml DCM wastreated with PyBOP (157 mg, 0.3 mmol) and HOAt (40 mg, 0.3 mmol) in thepresence of 2,4,6-collidine (67.5 μL, 0.5 mmol), yielding after work-upand purification 13.5 mg (27%) of the cyclic tetrapeptide with purity99% as determined by HPLC.

In this manner, compounds of formulae I-A through I-P were synthesized.The following Table 1 provides some data for the compounds of formulaeI-A through I-P.

TABLE 1 HPLC (Vydac C₁₈ 250 × 4.6 mm, 5 μm, 300 A, 1 mL/min, 45-75% B in15 min. A: 0.05% TFA/water, B: 0.038% TFA/82% Acetonitrile/water) MS(MH⁺)/MW Purity No. calculated t_(R) (min) (%, λ 214 nm) I-A503.45/502.6 4.79 99.35 I-B 503.47/502.6 5.96 99.00 I-C 517.49/516.65.26 99.00 I-D 503.25/502.6 5.10 98.12 I-E 503.20/502.6 5.83 97.15 I-F503.25/502.6 6.70 99.08 I-G 503.25/502.6 5.87 98.71 I-H 503.26/502.66.23 98.79 I-J 503.30/502.6 4.80 100.0 I-K 503.27/502.6 4.65 97.44 I-L 542.1/541.6 4.72 99.47 I-M  519.3/518.6 5.81 100.0 I-N  575.3/574.77.25 98.43 I-O  517.3/516.6 6.52 97.05 I-P MS + Na⁺ = 541.47, 9.15 99.00MS + K⁺ = 557.44/ (C-8 Kromasil col., 518.25 gradient 40-80% B in 15min)

Example 2 Therapeutic Efficacy of the Peptide as a 0.1% Ointment in theModel of Contact Sensitivity to Oxazolone in BALB/c Mice

The aim of this experiment was to verify the therapeutic action ofcompound I-A and its toxicity in a generally accepted animal model. Inthe experiment described below, the compound of formula I-A was appliedas a therapeutic preparation in the form of 0.1% wt/wt ointment based ona commonly used pharmaceutical vehicle, namely an ointment composed of50% vaseline and 50% lanoline. The usefulness of the preparation inreduction of the effector phase of the contact sensitivity to oxazolonein mice, in comparison with reference preparations such as tacrolimus(Protopic®) and pimecrolimus (Elidel®), widely used for treatment ofskin diseases, was studied.

Materials and Methods

Mice: BALB/c female mice, 8-10 week-old, delivered by the Institute ofLaboratory Medicine,

ódź, Poland, were used for the study. The mice were fed a commercial,pelleted food and water ad libitum. The local ethics committee approvedthe study.

Reagents: Water-in-oil cream and ointment were delivered by Nepentes.Cyclic tetrapeptide (compound I-A) was synthesized as described above.Protopic® (tacrolimus) was purchased from Astellas, Ireland; Elidel®(pimecrolimus) was purchased from Novartis; Hydrocortisonum®(hydrocortisone) was purchased from Aflofarm Farmacja Polska, Poland.DMSO was obtained from Fluka; oxazolone, acetone, Evans blue, Giemsa,May-Grünwald, and formalin were from Sigma.

Contact Sensitivity to Oxazolone:

The test was performed according to Noonan et al. (Int. Arch. AllergyAppl. Immunol., 1978, 56, 523-532), with some modifications. Mice wereshaved on the abdomen (2×2 cm area) and after 24 h 100 μl of 0.5%oxazolone in acetone was applied to the shaved area. The contactsensitivity reaction was elicited 5 days later by application of 50 μlof 1% oxazolone in acetone on both sides of the ears. Ear edema wasmeasured 48 h later using a spring caliper. The results were presentedas antigen-specific increase of ear thickness (i.e. the background (BG)ear thickness of mice was subtracted from the measured thickness).

Application of Compounds:

In the experiment shown, the compound of formula I-A was appliedtopically as a 0.1% ointment on both sides of the ears (total volume of50 μl per ear), at 24 h and 26 h after elicitation of the reaction withthe second dose of oxazolone. Reference compounds were used in a similarfashion in the form of commercially available preparations.

Determination of Lymph Node Cell Numbers:

Superficial parotid, mandibular and accessory mandibular lymph nodeswere isolated, homogenized by pressing against a stainless screen intophosphate buffered solution (PBS), washed twice and re-suspended in PBScontaining 0.2% Trypan blue. The total and nonviable cell numbers weredetermined using a light microscope and Bürker's hemocytometer. Micetreated only with the eliciting dose of antigen served as a backgroundcontrol.

Determination of Circulating Leukocyte Number and Blood Picture:

Mice were subjected to halothane anesthesia and bled from theretro-orbital plexus, followed by cervical dislocation. The number ofblood leukocytes was determined by dilution of blood in Türk's solutionand counting the cells in a hemocytometer. Blood smears were prepared onmicroscope glass, dried and stained with Giemsa and May-Grünwaldreagents. The smears were subsequently reviewed histologically. Thecirculating leukocyte numbers were presented per 1 mm³ and the bloodcell compositions as a percentage of a given cell type. Mice treatedonly with the eliciting dose of antigen served as a background control.

Histological Analysis:

The auricles were fixed in 4% formalin solution for 48 h, washed for 24h, dehydrated in an alcohol series and embedded in paraffin. Theparaffin blocks were sliced in a Micron HM310 microtome into 6 μmsections. The sections were stained with haematoxylin and eosin and withtoluidine blue. The histological analysis was performed in a NikonEclipse 801 light microscope. On the histological slides containingcross-sections of auricles, the morphometric estimations of neutrophils,macrophages, lymphocytes and mast cells in the perivascular andsubepithelial connective tissue were performed. The cells were countedon the area of 0.07 mm² at 400× magnification. Morphometric analysis wasdone with the aid of imagine software NIS-Elements (Nikon). For everyexamined group, 25 enumerations of neutrophils, macrophages, lymphocytesand mast cells were carried out.

Statistics:

The results are presented as mean values±standard error (SE).Brown-Forsyth's test was used to determine the homogeneity of variancebetween groups. When the variance was homogenous, analysis of variance(one-way ANOVA) was applied, followed by post hoc comparisons with theTukey's test to estimate the significance of the differences betweengroups. Nonparametric data were evaluated with the Kruskal-Wallis'analysis of variance, as indicated in the text. Significance wasdetermined at p<0.05. Statistical analysis was performed usingSTATISTICA 7 for Windows. The statistical analysis applies to all datashown in this description.

Results:

The data included in FIG. 1 show the therapeutic efficacy of compound offormula I-A (labeled “4B8M” in FIG. 1 and subsequent figures) and thereference preparations in mice with fully developed contact sensitivityreaction to oxazolone. The preparations were applied topically asdescribed in the Methods. FIG. 1 presents only antigen-specificincreases of the ear thickness (as a result of subtracting backgroundvalues measured in non-sensitized mice which were given only theeliciting dose of antigen). Compound I-A caused about 80% inhibition ofthe ear edema; Protopic® and Elidel® respectively caused about 30% and50% inhibition.

The intensity of inflammatory processes in the ears should correlatewith cell numbers in the draining lymph nodes. Therefore, inhibition ofinflammation should be associated with a decrease of cell number in thedraining lymph nodes. FIG. 2 shows that both the compound of formula I-Aas well as Elidel® decreased the numbers of lymphocytes in the draininglymph nodes to the level registered in non-sensitized mice. However, inmice treated with Protopic® the number of lymph node cells was similarto that in untreated mice.

FIG. 3 shows the proportions of viable and dead cells in the draininglymph nodes, expressed in percentages. The compound of formula I-Aexhibits a negligible toxic effect as compared to the control,non-treated mice. A higher toxic effect is caused by Elidel®, andProtopic® is exceptionally toxic with regard to lymph node cells.

Complementary information regarding the therapeutic efficacy of thepreparations may be derived from histological analysis of the cellnumber and composition in the inflamed auricles. FIG. 4 depicts thenumbers and participation of basic cell types involved in the localinflammatory process. In FIG. 4, Mast=mastocytes, L=lymphocytes,MØ=macrophages, Ne=neutrophils. The auricles from untreated mice (K+)are characterized by a high infiltration of neutrophils. Application ofthe compound of formula I-A almost entirely reversed the changesobserved in control mice (normalization of neutrophil number with someincrease in the macrophage content). Protopic®, in turn, caused somechanges in the proportion of respective cell types with no reduction ofthe total cell infiltrate. Elidel® caused a moderate diminution of thetotal cell number.

Example 3 The Toxicity of Compound I-A Versus that of CyclolinopeptideAgainst Human Blood Mononuclear Cells

For evaluation of toxicity of the compound of formula I-A, humanperipheral mononuclear blood cells (PBMC) were chosen. This fractionconsists of approximately 80% lymphocytes and 20% monocytes. As areference compound, cyclolinopeptide (CLA) was selected since thecompound of formula I-A shares a part of the sequence of CLA. CLAexhibits immunosuppressive properties comparable to that of cyclosporineA, but is less toxic.

Materials and Methods

The Cytotoxic Test:

Venous blood from a single donor was taken into heparinized syringes,diluted twice with phosphate buffered saline (PBS) and applied ontoLymphoprep® (Polfa, Kutno, Poland) (density of 1.077 g/ml). Aftercentrifugation at 1200×g for 20 min, the mononuclear cells from theinterphase were harvested and washed 3 times with PBS. The cells werere-suspended in a standard culture medium consisting of RPMI-1640medium, L-glutamine, sodium pyruvate, 2-mercaptoethanol, 100 μg/ml eachof streptomycin and penicillin, and 10% fetal calf serum. The cells weredistributed to in 96-well flat-bottom culture plates at density of2×10⁵/100 μl. The compounds (formula I-A and CLA) were initiallydissolved in DMSO (5 mg/500 μl) and subsequently in the culture medium.DMSO, appropriately diluted with the culture medium, was used as acontrol. After 24 h incubation in a cell culture incubator, cellviability was determined by a colorimetric method (Hansen et al., JImmunol Methods, 1989, 119, 203-210).

The results are shown in FIG. 5, presented as mean optical densityvalues from quadruplicate wells (cell cultures)±SE. As can be seen inFIG. 5, the compound of formula I-A (listed as “4B8M”) did not showappreciable toxicity in the concentration range of 10 to 100 μg/ml. CLA,on the other hand, showed a statistically significant cytotoxic effectat 40 μg/ml.

Example 4 Effect of Peptide on the Humoral Immune Response to SRBC InVivo

CBA female mice, 8-12 weeks old, were delivered by The Institute ofLaboratory Medicine,

ódź, Poland. The mice had free access to water and pelleted food. Thelocal ethics committee approved the study. Sheep erythrocytes (SRBC)were delivered by Wroclaw University of Life and Environmental Sciences,Poland, and maintained on RPMI-1640 medium.

The Primary Humoral Immune Response to SRBC In Vivo:

Mice were immunized with 0.2 ml of 5% SRBC suspension (0.5 ml of SRBCpellet re-suspended to a volume of 10 ml of 0.9% NaCl),intraperitonelly. After 4 days the number of antibody-forming cells(AFC) in the spleens was determined using an assay of local hemolysis inagar gel (per Mishell et al., J Exp Med, 1967, 126, 423-442). Theresults are presented in FIG. 6 as a mean value of 5 mice±standard errorand expressed as AFC number per 10⁶ of viable splenocytes.

Mice were immunized with SRBC as described above and after 2 h weregiven 10 or 100 μg of the compound of formula I-A. Cyclosporin A (CsA)served as a reference drug. The number of antibody-forming cells to SRBCwas measured after 4 days. As shown in FIG. 6, the compound of formulaIA was more inhibitory at both doses than CsA.

Example 5 Effect of Peptide on the Cellular Immune Response In Vivo toOvalbumin

Male CBA mice 8-12 weeks old were delivered by The Institute ofLaboratory Medicine,

ódź, Poland. The mice had free access to water and pelleted food.Ovoalbumin was from Sigma and the adjuvants from Difco.

Delayed Type Hypersensitivity (DTH) Test:

Mice were sensitized subcutaneously with 5 μg of ovalbumin (OVA)emulsified in Freund's complete adjuvant in the tail base. After 4 daysthe mice were challenged with 50 μg of OVA in Freund's incompleteadjuvant in the hind footpads. Following the next 24 hours, the footpadthickness was measured using a caliper. Controls (background responsemice) were not sensitized but received the challenging dose of OVA. Thecompound of formula I-A and the reference compound were administered tomice in two 100 μg intraperitoneal doses, 2 h before and 24 h after thesensitizing dose of antigen. The results, presented in FIG. 7 as a meanvalue of antigen-specific increase of footpad thickness measured in 5mice and expressed in DTH units (one DTH unit=10⁻² cm)±standard error,show that the compound of formula I-A, given in two doses, 2 h and 24 hafter immunization, inhibited the delayed-type hypersensitivity reactionto OVA. That suppressive action was stronger than those of CLA and CsA.

Example 6

Cyclic tetrapeptides were tested in vitro for their effects onphytohemagglutinin A (PHA)-induced proliferation of human peripheralblood mononuclear cells (PBMC) and for lipopolysaccharide (LPS)-inducedproduction of tumor necrosis factor alpha (TNF-α) production by wholeblood cell cultures at 1-100 μg/ml concentration range. Compounds werealso tested for cell toxicity at 1-100 μg/ml concentration range againsthuman PBMC.

Materials and Methods:

Reagents:

RPMI-1640 medium (Cibi/Life Technologies, UK), fetal calf serum (FCS,Gibco), DMSO, phytohemagglutinin A (PHA), lipopolysaccharide (LPS) fromE. coli strain 0111:B4 (Sigma),93-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT), SDSand DMF (Sigma). The culture medium consisted of RPMI-1640, 10% additionof FCS, L-glutamine, sodium pyruvate, 2-mercaptoetanol and antibiotics(streptomycin and penicillin). Cyclic tetrapeptides were initiallydissolved in DMSO (5 mg/ml), the dissolved in the culture medium to thedesired concentration.

Isolation of PBMC:

Venous blood was taken from a single donor (a male, 62-years old) intoheparinized syringes and diluted twice with phosphate-buffered saline(PBS). PBMC were isolated by centrifugation on Ficoll-uropoline gradient(density 1.077 g/ml) (Lymphoprep; PAA Laboratories), at 800×g for 20 minat 4° C. The interphase cells, consisting of lymphocytes (20%) andmonocytes (80%) were then washed three times with Hanks' medium andre-suspended in the culture medium at density of 2×10⁶ cells/ml.

The Proliferative Response of PBMC to PHA:

The isolated PBMC were distributed into 96-well flat-bottom plates in100 μl aliquots (2×10⁵ cells/well). PHA was used at concentration of 5μg/ml. The compounds were tested at concentrations of 1, 10 and 100μg/ml. DMSO at appropriate dilutions served as control. After a four-dayincubation in a cell culture incubator, the proliferative response ofcells was determined by the colorimetric MTT method (Hansen et al., J.Immunol. Methods, 1989, pp. 203-210). The data are presented as a meanOD value from quadriplicate wells±standard error (SE). The cultures“Control (−)” contained no mitogen (PHA). The cultures “Control (PHA)”contained PHA but not cyclic tetrapeptides.

Toxicity Test:

PBMC, at a density of 2×10⁵/100 μl/well, re-suspended in the culturemedium, were cultured for 24 h in a cell culture incubator with thecyclic tetrapeptides at concentrations of 1, 10 and 100 μg/mlconcentrations. Cell survival was determined by the MTT colorimetricmethod (Hansen et al., J. Immunol. Methods, 1989, pp. 203-210). The dataare presented as a mean OD value from quadriplicate wells±standard error(SE). The cultures “Control (−)” contained only cells in the culturemedium.

The Determination of TNF Alpha Activity (Per Espevik et al., J. Immunol.Methods, 95 (1986):99-103):

Human whole blood was diluted 10-fold with RPMI-1640 medium anddistributed to 24-well culture plates in 1 ml aliquots. LPS was added tothe culture at a concentration of 1 μg/ml. The studied peptides wereused at concentrations of 1, 10 and 100 μg/ml. After overnightincubation, the supernatants were harvested and frozen at −20° C. untilcytokine determination. TNF-α activity was determined using a bioassay.Briefly, WEHI 164.13 cells (ATCC CRL 1751) were seeded at aconcentration of 2×10⁴ cells/well in quadriplicate. Increasing dilutionsof the assayed supernatant were mixed with the target cells in thepresence of actinomycin D (1 μg/ml). After 20 h of incubation, MTT wasadded into the wells, and the cultures were incubated for an additional4 h. Next, a lysing buffer (20% SDS with 50% DMF, pH 4.7) was added andthe optical density at 550 nm with the reference wavelength of 630 nm ina Dynatech 5000 spectrophotometer was measured after 24 h. The detectionlimit of the assay was about 2.5 pg/ml. One unit of TNF-α activity wasdefined as an inverse of supernatant dilution where 50% cell death tookplace. The cultures labeled “Control (−)” contained no LPS. The cultureslabeled “Control (LPS)” contained LPS and none of the studied compounds.Statistical analysis was not applied since the data derive from singlecultures (wells).

Colorimetric MTT Assay for Cell Growth and Kill:

The assay was performed per Hansen et al., J. Immunol. Methods, 1989,119 pp. 203-210. Briefly, 25 μl of MTT (5 mg/ml) stock solution wasadded per well at the end of cell incubation and the plates wereincubated for 3 h in a cell culture incubator. Then, 100 μl of theextraction buffer (20% SDS with 50% DMF, pH 4.7) was added. Afteradditional overnight incubation, the optical density was measured at 550nm (Dynatech 5000).

Where applicable, results are presented as mean values±standard error(SE). Brown-Forsyth's test was used to determine the homogeneity ofvariance between groups. When the variance was homogenous, analysis ofvariance (one-way ANOVA) was applied, followed by post hoc comparisonswith the Tukey's test to estimate the significance of the differencesbetween groups. Significance was determined at P<0.05. Statisticalanalysis was performed using STATISTICA 6.1 for Windows.

Results

Effects of the Compounds on Survival of PBMC:

The effects of the peptides on PMBC survival in 24 h culture arepresented in FIGS. 8A and 8B. Peptide 4B8M (compound I-A) was includedas a reference compound. Appropriate dilutions of DMSO were added tocontrol cultures. The results showed no signs of toxicity of thecompounds in the studied concentration range. In the figures,P01=compound I-D, P02=compound I-E, P03=compound I-F, P04=compound I-G,P05=compound I-H, P06=compound I-J, P07=compound I-K, P08=compound I-L,P10a=compound I-M, P10b=compound I-N, and P11=compound I-O.

FIGS. 8A and 8B show the effects of the tested peptides on the survivalof PBMC. FIG. 8A: Statistics (all comparisons vs. DMSO at appropriatedilutions): 100 μg/ml: 4B8M NS (P=0.9999); P01 NS (P=1.0000); P02 NS(P=1.0000); P03 NS (P=1.0000); P04 NS (P=0.9047); P05 NS (P=1.0000); P06NS (P=0.9999); P07 NS (P=1.0000); 4B8M NS (P=1.0000); P01 NS (P=1.0000);P02 NS (P=1.0000); P03 NS (P=1.0000); P04 NS (P=0.9999); P05 NS(P=1.0000); P06 NS (P=1.0000); P07 NS (P=1.0000); 1 μg/ml: 4B8M NS(P=1.0000); P01 NS (P=1.0000); P02 NS (P=1.0000); P03 NS (P=1.0000); P04NS (P=1.0000); P05 NS (P=1.0000); P06 NS (P=0.8253); P07 NS (P=1.0000)(ANOVA). FIG. 8B: Statistics (all comparisons vs. DMSO at appropriatedilutions): 100 μg/ml: 4B8M NS (P=0.0669); P08 NS (P=0.9957); P10b NS(P=1.0000); 10 μg/ml: 4B8M NS (P=0.9999); P08 NS (P=1.0000); P10b NS(P=1.0000); 1 μg/ml: 4B8M NS (P=0.3176); P08 NS (P=0.9999); P10b NS(P=1.0000) (ANOVA).

Effects of the Peptides on PHA-Induced PMBC Proliferation:

The effects of the peptides on the proliferative response of PMBC arepresented in FIGS. 9A and 9B. Peptide 4B8M was included as a referencecompound. Appropriate dilutions of DMSO were added to control cultures.

FIG. 9A: Effects of the peptides on PHA-induced PBMC proliferation:Statistics (all comparisons vs. DMSO at appropriate dilutions): 1 μg/ml:4B8M NS (P=0.9995); P01 NS (P=1.0000); P02 NS (P=1.0000); P03 NS(P=1.0000); P04 NS (P=0.9047); P05 NS (P=0.5198); P06 NS (P=1.0000); P07NS (P=0.1445); 10 μg/ml: 4B8M NS (P=1.0000); P01 NS (P=0.9999); P02 NS(P=1.0000); P03 NS (P=0.9930); P04 NS (P=0.4297); P05 NS (P=1.0000); P06NS (P=1.0000); P07 NS (P=0.8647); 100 μg/ml: 4B8M NS (P=1.0000); P01 NS(P=1.0000); P02 NS (P=0.9982); P03 P=0.0001; P04 NS (P=0.9970); P05 NS(P=0.2037); P06 NS (P=0.1257); P07 NS (P=1.0000) (ANOVA).

FIG. 9B: Effects of the peptides on PHA-induced PBMC proliferation:Statistics (all comparisons vs. DMSO at appropriate dilutions): 1 μg/ml:4B8M NS (P=0.9919); P08 NS (P=0.9999); P10b NS (P=1.0000); 10 μg/ml:4B8M NS (P=1.0000); P08 NS (P=1.0000); P10b NS (P=0.2763); 100 μg/ml:4B8M NS (P=0.4941); P08 NS (P=1.0000); P10b NS (P=0.9933) (ANOVA).

Effects of the Peptides on LPS-Induced TNF-α Production in Whole BloodCell Cultures:

The effects of the peptides on LPS-induced TNF-α production in wholeblood cell cultures are presented in Tables 2A and 2B. Peptide 4B8M wasincluded as a reference compound. Appropriate dilutions of DMSO wereadded to control cultures.

TABLE 2A Effects of peptides on LPS-induced TNF-α production compoundconcentration TNF-α % inhibition Compound (μg/ml) (pg/ml) vs. DMSOControl (—) — 92 — Control (LPS) — 6668 — DMSO 1 8167 — Only 10 8006 —100 196 — 4B8M 1 7364 9.8 (compound I-A) 10 7176 10.4 100 178 9.2 P01 17016 14.1 (compound I-D) 10 5757 28.1 100 141 28.1 P02 1 6614 19.0(compound I-E) 10 6346 20.7 100 176 10.2 P03 1 6052 25.9 (compound I-F)10 5222 34.8 100 76 61.2 P04 1 5463 33.1 (compound I-G) 10 5302 33.8 10058 70.4 P05 1 7123 12.8 (compound I-H) 10 7203 10.0 100 110 43.9 P06 18033 1.6 (compound I-J) 10 7658 4.4 100 203 0.0 P07 1 6453 21.0(compound I-L) 10 7284 9.0 100 163 16.8

TABLE 2B Effects of the peptides on LPS-induced TNF alpha productioncompound % of concentration TNF alpha inhibition Compound (μg/ml)(pg/ml) vs. DMSO Control (—) — 223 — Control (LPS) — 4260 — DMSO 1 4550— Only 10 4351 — 100 854 — 4B8M 1 4097 10.0 (compound I-A) 10 4416 — 100979 — P08 1 4039 11.2 (compound I-M) 10 3363 22.7 100 269 68.5 P10b 13792 16.7 (compound I-O) 10 3739 14.0 100 282 67.0

Example 7 Inhibitory Effect of Compound I-A on TolueneDiisocyanate-Induced Ear Inflammation in Mice

The efficacy of the compound I-A in suppressing ear inflammation inBALB/c mice which was induced with toluene diisocyanate (TDI).Commercially available Protopic® (tacrolimus) and Elidel® (pimecrolimus)served as reference drugs.

Materials and Methods

Mice:

BALB/c female mice, 8-10 weeks old, were delivered by the Institute ofLaboratory Medicine,

ódź, Poland. The mice were fed a commercial, pelleted food and water adlibitum. The local ethics committee approved the study.

Reagents.

Compound I-A was synthesized as described above; Protopic® (tacrolimus)was from Astellas, Ireland; Elidel® (pimecrolimus) from Novartis; DMSOfrom Fluka; TDI, acetone, Evans blue, Trypan blue, Giemsa, May-Grünwald,haematoxylin, eosin, toluidine blue and formalin were from Sigma.

Immune Response to TDI.

The test was performed according to Yamamoto, Eur. J. Pharmacol., 2006,550, 166-172, with minor modifications. Mice were shaved on the abdomen(2×2 cm area) and after 24 h 100 μl of 3% TDI in acetone was appliedthrough 3 consecutive days. After 14 days the reaction was elicited byapplication of 50 μl of 0.3% TDI on both sides of the ears. Theprocedure was repeated 5 times every 3 days. Ear thickness was measuredusing a spring caliper (Mitutoyo) 5 h and 24 h after each challenge withTDI.

Application of Compounds.

Compound I-A was applied topically in the form of 0.1% ointment on bothsides of the ears (total volume of 100 μl−50 μl per ear), one hour aftereach challenge with TDI. The reference drugs were applied in a similarway.

Determination of Lymph Node Cell Numbers.

Superficial parotid, mandibular and accessory mandibular lymph nodeswere isolated, homogenized by pressing against a stainless screen intoPBS, washed 2× with PBS and re-suspended in PBS containing 0.2% Trypanblue. The total and nonviable cell numbers were counted using a lightmicroscope and Bürker's hemocytometer.

Determination of Circulating Leukocyte Number and Blood Picture.

Mice were subjected to halothane anesthesia and bled from theretro-orbital plexus, followed by the cervical dislocation. The numberof blood leukocytes was determined by dilution of blood in Türk'ssolution and counting the cells in a hemocytometer. Blood smears wereprepared on microscope glass, dried and stained with Giemsa andMay-Grünwald reagents. The smears were subsequently reviewedhistologically. The cell numbers were presented per 1 μl and the bloodcell compositions as a percentage of a given cell type.

Evans Blue Test.

Mice were given 1 mg of Evans blue in 0.2 ml of 0.9% NaCl,intravenously. After 30 min mice were sacrificed, the ears were cut off,weighed and immersed in 50 μl of 1M KOH for 18 h at 37° C. The dye wasextracted from the ears using 450 μl of 0.2 M phosphate acid and acetone(5:13 ratio). The samples were centrifuged at 3,000 rpm for 15 min. Theoptical densities (OD) of the supernatants were measured at 630 nm. Theamount of Evans blue (μg/ml) was determined based on a standard curve.The results were presented as the amount of Evans blue per 100 mg of wettissue. Mice treated only with the eliciting dose of antigen served as abackground control.

Histological Analysis.

The auricles were fixed in 4% formalin solution for 48 h, washed for 24h, dehydrated in an alcohol series and embedded in paraffin. Theparaffin blocks were sliced in a Micron HM310 microtome into 6 μmsections. The sections were stained with haematoxylin and eosin and withtoluidine blue. Histological analysis was performed using a NikonEclipse 801light microscope. Morphometric estimations of neutrophils,macrophages, lymphocytes and mast cells in the perivascular andsubepithelial connective tissue were performed on the histologicalslides containing cross-sections of auricles. Cells were counted on anarea of 0.07 mm² at 400× magnification. Morphometric analysis wasperformed using an imagine software NIS-Elements (Nikon). For everypreparation examined, 25 enumerations of neutrophils, macrophages,lymphocytes and mast cells were carried out.

Statistics.

The results in FIGS. 10A and 10B are presented as mean values±standarderror (SE). Brown-Forsyth's test was used to determine the homogeneityof variance between the groups. When the variance was homogenous,analysis of variance (one-way ANOVA) was applied, followed by post hoccomparisons with the Tukey's test to estimate the significance of thedifferences between groups. Nonparametric data were evaluated with theKruskal-Wallis' analysis of variance, as indicated in the text.Significance was determined at P<0.05. Statistical analysis wasperformed using STATISTICA 7 for Windows.

Results

Effects of the Compounds on the Ear Thickness.

The humoral immune response to TDI was elicited as described in theMethods. Mice were treated topically with the tetrapeptide (formula I-A,labeled 4B8M in FIGS. 10A and 10B) and the reference compounds one hourafter each challenge with antigen. The effects of the treatments arepresented in FIGS. 10A and 10B, which show ear thickness measure 5 h(FIG. 10A) and 24 h (FIG. 10B) after administration on the day of thetest indicated in the figure. Control responses to TDI graduallyelevated after each antigen challenge (best seen in the 5 hmeasurement). The results showed differentiated efficacy of thecompounds in reducing the ear swelling.

FIG. 10A: Day 14: Control vs 4B8M P=0.0005; Control vs Protopic®P=0.0002; Control vs Elidel® P=0.0152; 4B8M vs Protopic® NS; 4B8M vsElidel® NS; Day 17: Control vs 4B8M P=0.0001; Control vs Protopic®P=0.0001; Control vs Elidel® P=0.0377; 4B8M vs Protopic® NS; 4B8M vsElidel® P=0.0002; Day 20: Control vs 4B8M P=0.0001; Control vs Protopic®P=0.0001; Control vs Elidel® NS; 4B8M vs Protopic® NS; 4B8M vs Elidel®P=0.0001; Day 23: Control vs 4B8M P=0.0001; Control vs Protopic®P=0.0001; Control vs Elidel® NS; 4B8M vs Protopic® NS; 4B8M vs Elidel®P=0.0001; Day 27: Control vs 4B8M P=0.0001; Control vs Protopic®P=0.0001; Control vs Elidel® P=0.0001; 4B8M vs Protopic® NS; 4B8M vsElidel® P=0.0001 (ANOVA). FIG. 10B: Day 14: Control vs 4B8M P=0.0001;Control vs Protopic® P=0.0001; Control vs Elidel® P=0.0004; 4B8M vsProtopic® NS; 4B8M vs Elidel® NS; Day 17: Control vs 4B8M P=0.0001;Control vs Protopic® P=0.0023; Control vs Elidel® P=0.0004; 4B8M vsProtopic® NS; 4B8M vs Elidel® NS; Day 20: Control vs 4B8M P=0.0006;Control vs Protopic® P=0.0003; Control vs Elidel® P=0.0156; 4B8M vsProtopic® NS; 4B8M vs Elidel® NS; Day 23: Control vs 4B8M P=0.0001;Control vs Protopic® P=0.0001; Control vs Elidel® P=0.0039; 4B8M vsProtopic® NS; 4B8M vs Elidel® P=0.0027; Day 27: Control vs 4B8MP=0.0001; Control vs Protopic® P=0.0001; Control vs Elidel® P=0.0023;4B8M vs Protopic® NS; 4B8M vs Elidel® P=0.0016 (ANOVA).

Effects of the Compounds on Permeability of Skin Vessels:

The permeability of capillary blood vessels is presented in FIG. 11,which shows the permeability of capillary blood vessels in the Evansblue test. The procedure, as described aboved, was performed 24 h afterthe fifth challenge with TDI (on day 28). As shown in the FIG. 11, therates of blood vessel permeability were strictly correlated with theeffects of the compounds on ear thickness in the respective mousegroups. Statistics: BG vs Control P=0.0248; Control vs 4B8M (I-A)P=0.030; Control vs Protopic® NS; Control vs Elidel® NS; 4B8M (I-A) vsProtopic® NS; 4B8M (I-A) vs Elidel® NS (ANOVA).

Effects of the Compounds on Number of Cells in Draining Lymph Nodes:

FIG. 12 shows the total number of cells in the draining lymph nodes. Asshown in FIG. 12, the treatment of mice with compound I-A resulted in areduction of the lymph node cell numbers almost to the background levels(non-sensitized mice). Statistics: BG vs Control P=0.0001; Control vs4B8M (I-A) P=0.0001; Control vs Protopic® NS; Control vs Elidel® NS;4B8M (I-A) vs Protopic® P=0.0183; 4B8M (I-A) vs Elidel® P=0.0001 (ANOVAof Kruskal-Wallis).

Effects of the Compounds on the Numbers of Circulating Leukocytes.

FIG. 13 shows the effects of the compounds on the numbers of circulatingleukocytes; the application of the studied preparations lowered thenumbers of circulating leukocytes to the levels observed in control,unsensitized mice. Statistics: BG vs Control P=0.0001; Control vs 4B8M(I-A) P=0.0001; Control vs Protopic® P=0.0001; Control vs Elidel®P=0.0001; 4B8M (I-A) vs Protopic® NS; 4B8M (I-A) vs Elidel® NS (ANOVA).

Effects of Compounds on Blood Cell Composition:

The blood composition in control mice with fully developed reaction toTDI was characterized by an increased content of neutrophils andeosinophils compared to control, background mice (FIG. 14, which shows abreakdown of the types of leukocytes in each case). The blood picturewas normalized upon application of 4B8M (I-A) (a reduction of neutrophiland eosinophil contents) but not following administration of Protopic®or Elidel®. Statistics: Bands (B): BG vs Control NS; Control vs 4B8M(I-A) NS; Control vs Protopic® P=0.0500; Control vs Elidel® P=0.0500;4B8M (I-A) vs Protopic® NS; 4B8M vs Elidel® NS (ANOVA ofKruskal-Wallis); Segments (S): BG vs Control P=0.0131; Control vs 4B8M(I-A) NS; Control vs Protopic® NS; Control vs Elidel® NS; 4B8M (I-A) vsProtopic® P=0.0163; 4B8M (I-A) vs Elidel® NS (ANOVA of Kruskal-Wallis);Eosinophils (E): BG vs Control P=0.0001; Control vs 4B8M (I-A) P=0.0001;Control vs Protopic® NS; Control vs Elidel® NS; 4B8M (I-A) vs Protopic®NS; 4B8M vs Elidel® P=0.0146 (ANOVA of Kruskal-Wallis); Lymphocytes (L):BG vs Control P=0.0001; Control vs 4B8M (I-A) P=0.0043; Control vsProtopic® NS; Control vs Elidel® NS; 4B8M (I-A) vs Protopic® P=0.0345;4B8M (I-A) vs Elidel® NS (ANOVA of Kruskal-Wallis).

The Effects of the Compounds on Cell Composition in the Auricles:

FIG. 15 provides morphometric data on the number and composition ofcells in the auricles. The composition of cell types within the auriclesdiffered among the studied mouse groups is presented in FIG. 15. Thepredominant, residing cell types in control non-sensitized mice aremastocytes and neutrofils (10 and 5 cells per the analyzed area,respectively). In sensitized, control mice, untreated with thetherapeutics, the number of mastocytes increased twofold and neutrophilsalmost 5-fold (20 and 23 cells, respectively). Protopic® and CompoundI-A were effective in reducing the cell numbers to 14 and 14.8.Statistics: Ne (neutrophils): BG vs Control P=0.0001; Control vs 4B8M(I-A) P=0.0151; Control vs Protopic® P=0.0001; Control vs Elidel® NS;4B8M vs Protopic® NS; 4B8M (I-A) vs Elidel® P=0.0003 (ANOVA ofKruskal-Wallis); MØ (macrophages): BG vs Control P=0.0144; Control vs4B8M (I-A) NS; Control vs Protopic® NS; Control vs Elidel® P=0.0255;4B8M vs Protopic® NS; 4B8M (I-A) vs Elidel® P=0.0031 (ANOVA ofKruskal-Wallis); L (lymphocytes): BG vs Control NS; Control vs 4B8M(I-A) NS; Control vs Protopic® NS; Control vs Elidel® P=0.0023; 4B8M vsProtopic® NS; 4B8M (I-A) vs Elidel® P=0.0001 (Anova of Kruskal-Wallis);Mast (mastocytes): BG vs Control P=0.0001; Control vs 4B8M (I-A) NS;Control vs Protopic® P=0.0001; Control vs Elidel® NS; 4B8M (I-A) vsProtopic® NS; 4B8M (I-A) vs Elidel® P=0.0001 (ANOVA of Kruskal-Wallis).

Example 8 In Vitro Tests of Compounds I-B and I-C Methods

Proliferative Response of Splenocytes to Concanavalin A (ConA):

Spleens were pressed against a plastic screen into 0.83% NH₄Cl solutionto lyse erythrocytes (5 min incubation at room temperature). The cellswere then washed twice with Hanks' medium, passed through glass woolcolumn to remove debris, and re-suspended in the culture medium,referred to below as the “culture medium”, consisting of RPMI-1640,supplemented with 10% of fetal calf serum, L-glutamine, sodium pyruvate,2-mercaptoethanol, streptomycin and penicillin (100 μg/ml). The cellswere then distributed into 96-well flat-bottom tissue culture plates(Nunc) at a density of 2×10⁵ cells/100 μl/well. Con A (2.5 μg/ml) wasadded to induce cell proliferation. The compounds were added to thecultures at doses of 1-100 μg/ml. After a three-day incubation, cellproliferation was determined using the colorimetric MTT method (Hansen MB, J Immunol Methods, 1989, 119, 203-210). The results were presented asthe mean optical density (OD) at 550 nm±SE from quadriplicatedeterminations (wells).

Secondary Humoral Immune Response In Vitro to Sheep Erythrocytes (SRBC):

Mice were sensitized intraperitoneally with 0.2 ml of 5% (v/v) SRBCsuspension. After four days spleens from these mice were isolated andsingle cell suspensions prepared by homogenization in PBS solution.After washing the cells in PBS by centrifugation, the cells werere-suspended in the culture medium at a density of 5×10⁶ cells/ml. Thecells were subsequently distributed to 24-well culture plates in 1 mlaliquots and 0.05 ml of 0.005% SRBC was added as the antigen to eachwell. The compounds were added to the cultures at the beginning of thefour-day incubation period at concentration ranges of 1-100 μg/ml. Thenumber of antibody-forming cells (AFC) in the cultures was determinedusing a method of local hemolysis in agar gel according to Mishell etal., J Exp Med, 1967, 126, 423-442.

Toxicity Test:

Splenocytes, at a density of 2×10⁵ cells/100 μl/well, re-suspended inthe culture medium, were cultured for 24 h in a cell culture incubatorwith the compounds (1-100 μg/ml). Cell survival was determined by MTTcolorimetric method. The results were presented as mean optical density(OD) at 550 nm from 4 wells. The viability of cells in respectivecompound concentrations was compared to appropriate DMSO control groups(100% survival), corresponding to respective compound concentrations.

Results

At 50-100 μg/ml concentrations, compound I-C showed a strong inhibitoryeffect on concanavalin A-induced mouse splenocyte proliferation. At 100μg/m, this compound showed 70% toxicity to splenocytes. At 10 μg/m and100 μg/m, compound I-C showed 33% and 80% suppression, respectively, inthe model of in vitro humoral immune response to SRBC in mousesplenocyte cultures. In the model of delayed type hypersensitivity toovalbumin, compound I-C showed 26.9% inhibition at the dose of 100 μg,compared to 72.7% inhibition by compound I-A.

At 100 μg/ml concentration, compound I-B demonstrated a weakantiproliferative effect on concanavalin A-induced splenocyteproliferation; no such effect was observed at lower concentrations.Compound I-B had 30% toxicity at this concentration.

Although the foregoing invention has been described in some detail forpurposes of illustration, it will be readily apparent to one skilled inthe art that changes and modifications may be made without departingfrom the scope of the invention described herein.

1. A compound having the formula I:

wherein k, m, n and p are each independently 0, 1 or 2, provided that atleast one of k, m, n and p is not 0; R and R′ are each independentlyselected from H and C₁₋₃ alkyl, or, when taken together, R and R′ are—CR¹R^(1′)—X—CH₂—, wherein CR¹R^(1′) is attached to the backbonenitrogen, R¹ and R^(1′) are each independently selected from H and C₁₋₃alkyl, and X is selected from —CH₂—, —CH₂CH₂—, —CH(OH)—, —O—, —S— and—NH—; R″ and R′″ are each independently selected from H and C₁₋₃ alkyl,or, when taken together, R″ and R′″ are —CR²R^(2′)—X′—CH₂—, whereinCR²R^(2′) is attached to the backbone nitrogen, R² and R^(2′) are eachindependently selected from H and C₁₋₃ alkyl, and X′ is selected from—CH₂—, —CH₂CH₂—, —CH(OH)—, —O—, —S— and —NH—; and R³ and R⁴ are eachindependently selected from aryl, substituted aryl, heteroaryl andsubstituted heteroaryl; or a pharmaceutically acceptable salt thereof.2-243. (canceled)
 244. A compound according to claim 1 wherein at leastone of R³ and R⁴ is selected from the group consisting of phenyl,4-hydroxyphenyl, 4-t-butoxyphenyl and 2-indolyl.
 245. A compoundaccording to claim 1 wherein one of R³ and R⁴ is phenyl and the other ofR³ and R⁴ is selected from the group consisting of phenyl,4-hydroxyphenyl, 4-t-butoxyphenyl, and 2-indolyl.
 246. A compoundaccording to claim 1 wherein at least one of the following is true: (a)one of k, m, n and p is 1, and the remainder of k, m, n and p are 0; (b)two of k, m, n and p are 1, and the remainder of k, m, n and p are 0;(c) at least one of k and m is not 0; (d) at least one of n and p is not0; (e) at least one of k and m is not 0 and at least one of n and p isnot 0; (f) both k and n are 0; (g) both k and n are 0, one of m and p is0, and the other of m and p is 1; (h) both k and n are 0 and both m andp are
 1. 247. A compound according to claim 1 wherein at least one ofthe following is true: (a) R and R′ are taken together to form —(CH₂)₃—;(b) R″ and R′″ are taken together to form —(CH₂)₃—; (c) R and R′ aretaken together to form —(CH₂)₄—; (d) R″ and R′″ are taken together toform —(CH₂)₄—; (e) R and R′ are taken together to form —CH₂—CH(OH)—CH₂—(f) R″ and R′″ are taken together to form —CH₂—CH(OH)—CH₂—.
 248. Acompound according to claim 1 wherein the compound is selected from thegroup consisting of:


249. A compound according to claim 248 which is selected from the groupconsisting of:


250. A pharmaceutical composition comprising a compound having theformula I:

wherein k, m, n and p are each independently 0, 1 or 2, provided that atleast one of k, m, n and p is not 0; R and R′ are each independentlyselected from H and C₁₋₃ alkyl, or, when taken together, R and R′ are—CR¹R^(1′)—X—CH₂—, wherein CR¹R^(1′) is attached to the backbonenitrogen, R¹ and R^(1′) are each independently selected from H and C₁₋₃alkyl, and X is selected from —CH₂—, —CH₂CH₂—, —CH(OH)—, —O—, —S— and—NH—; R″ and R′″ are each independently selected from H and C₁₋₃ alkyl,or, when taken together, R″ and R′″ are —CR²R^(2′)—X′—CH₂—, whereinCR²R^(2′) is attached to the backbone nitrogen, R² and R^(2′) are eachindependently selected from H and C₁₋₃ alkyl, and X′ is selected from—CH₂—, —CH₂CH₂—, —CH(OH)—, —O—, —S— and —NH—; and R³ and R⁴ are eachindependently selected from aryl, substituted aryl, heteroaryl andsubstituted heteroaryl; or a pharmaceutically acceptable salt thereof,and a pharmaceutically acceptable carrier, excipient or diluenttherefor.
 251. A pharmaceutical composition according to claim 250wherein the compound is selected from the group consisting of


252. A pharmaceutical composition according to claim 251 wherein thecompound is selected from the group consisting of:


253. A pharmaceutical composition according to claim 250 wherein atleast one of the following is true of the compound of formula I: (a) Rand R′ are taken together and the carbon at which R′ is attached hasabsolute (S)-stereochemistry; (b) R″ and R′″ are taken together and thecarbon at which R′″ is attached has absolute (S)-stereochemistry.
 254. Apharmaceutical composition according to claim 250 wherein the carbon towhich —CH₂—R³ is attached has absolute (S)-stereochemistry and thecarbon which —CH₂—R⁴ is attached has absolute (S)-stereochemistry. 255.A pharmaceutical composition according to claim 250 wherein at least oneof the carbons to which R′, R′″, —CH₂R³ and —CH₂R⁴ are respectivelyattached has absolute (R)-stereochemistry.
 256. A method of suppressingimmune response in a patient, or of treating or preventing animmune-mediated disease or condition in a patient, comprisingadministering to a patient in need thereof an efficacious amount of acompound of formula I:

wherein k, m, n and p are each independently 0, 1 or 2, provided that atleast one of k, m, n and p is not 0; R and R′ are each independentlyselected from H and C₁₋₃ alkyl, or, when taken together, R and R′ are—CR¹R^(1′)—X—CH₂—, wherein CR¹R^(1′) is attached to the backbonenitrogen, R¹ and R^(1′) are each independently selected from H and C₁₋₃alkyl, and X is selected from —CH₂—, —CH₂CH₂—, —CH(OH)—, —O—, —S— and—NH—; R″ and R′″ are each independently selected from H and C₁₋₃ alkyl,or, when taken together, R″ and R′″ are —CR²R^(2′)—X′—CH₂—, whereinCR²R^(2′) is attached to the backbone nitrogen, R² and R^(2′) are eachindependently selected from H and C₁₋₃ alkyl, and X′ is selected from—CH₂—, —CH₂CH₂—, —CH(OH)—, —O—, —S— and —NH—; and R³ and R⁴ are eachindependently selected from aryl, substituted aryl, heteroaryl andsubstituted heteroaryl; or a pharmaceutically acceptable salt thereof.257. A method according to claim 256 wherein the immune-mediated diseaseor condition is selected from the group consisting of (a) the groupconsisting of auto-immune diseases, inflammation processes, transplantrejection, and allergic reactions, (b) the group consisting ofPsoriasis, lichen planus and other papulosquamous disorders, (c) thegroup consisting of eczema and dermatitis (including eczema, atopiceczema, seborrheic dermatitis, and pompholyx), (d) a skin reaction tolight, (e) the group consisting of non-specific skin irritation andinsect bite, (f) a urticaria, and (g) the group consisting of rheumatoidarthritis (both autoimmune and elicited by infection), Crohn's disease,inflammatory bowel disease, irritable bowel syndrome, aneurodegenerative disease (e.g. multiple sclerosis), Graft-versus-Hostreaction, severe psoriasis and atopic dermatitis.
 258. A compoundaccording to claim 1 wherein at least one of the following is true: (a)R and R′ are taken together and the carbon at which R′ is attached hasabsolute (S)-stereochemistry; (b) R″ and R′″ are taken together and thecarbon at which R′″ is attached has absolute (S)-stereochemistry.
 259. Acompound according to claim 1 wherein the carbon to which —CH₂—R³ isattached has absolute (S)-stereochemistry and the carbon which —CH₂—R⁴is attached has absolute (S)-stereochemistry.
 260. A compound accordingto claim 1 wherein one or more amino groups in the compound of Formula Iare in protected form.
 261. A compound according to claim 1 wherein atleast one of the carbons to which R′, R′″, —CH₂R³ and —CH₂R⁴ arerespectively attached has absolute (R)-stereochemistry.
 262. Thecompound according to claim 1 which is the compound of formula I-A or apharmaceutically acceptable salt thereof.